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

rAvis: an R-package for downloading information stored in Proyecto AVIS, a citizen science bird project.

PubMed

Varela, Sara; González-Hernández, Javier; Casabella, Eduardo; Barrientos, Rafael

2014-01-01

Citizen science projects store an enormous amount of information about species distribution, diversity and characteristics. Researchers are now beginning to make use of this rich collection of data. However, access to these databases is not always straightforward. Apart from the largest and international projects, citizen science repositories often lack specific Application Programming Interfaces (APIs) to connect them to the scientific environments. Thus, it is necessary to develop simple routines to allow researchers to take advantage of the information collected by smaller citizen science projects, for instance, programming specific packages to connect them to popular scientific environments (like R). Here, we present rAvis, an R-package to connect R-users with Proyecto AVIS (http://proyectoavis.com), a Spanish citizen science project with more than 82,000 bird observation records. We develop several functions to explore the database, to plot the geographic distribution of the species occurrences, and to generate personal queries to the database about species occurrences (number of individuals, distribution, etc.) and birdwatcher observations (number of species recorded by each collaborator, UTMs visited, etc.). This new R-package will allow scientists to access this database and to exploit the information generated by Spanish birdwatchers over the last 40 years.

rAvis: An R-Package for Downloading Information Stored in Proyecto AVIS, a Citizen Science Bird Project

PubMed Central

Varela, Sara; González-Hernández, Javier; Casabella, Eduardo; Barrientos, Rafael

2014-01-01

Citizen science projects store an enormous amount of information about species distribution, diversity and characteristics. Researchers are now beginning to make use of this rich collection of data. However, access to these databases is not always straightforward. Apart from the largest and international projects, citizen science repositories often lack specific Application Programming Interfaces (APIs) to connect them to the scientific environments. Thus, it is necessary to develop simple routines to allow researchers to take advantage of the information collected by smaller citizen science projects, for instance, programming specific packages to connect them to popular scientific environments (like R). Here, we present rAvis, an R-package to connect R-users with Proyecto AVIS (http://proyectoavis.com), a Spanish citizen science project with more than 82,000 bird observation records. We develop several functions to explore the database, to plot the geographic distribution of the species occurrences, and to generate personal queries to the database about species occurrences (number of individuals, distribution, etc.) and birdwatcher observations (number of species recorded by each collaborator, UTMs visited, etc.). This new R-package will allow scientists to access this database and to exploit the information generated by Spanish birdwatchers over the last 40 years. PMID:24626233

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.

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.

TrAVis to Enhance Online Tutoring and Learning Activities: Real-Time Visualization of Students Tracking Data

ERIC Educational Resources Information Center

May, Madeth; George, Sebastien; Prevot, Patrick

2011-01-01

Purpose: This paper presents a part of our research work that places an emphasis on Tracking Data Analysis and Visualization (TrAVis) tools, a web-based system, designed to enhance online tutoring and learning activities, supported by computer-mediated communication (CMC) tools. TrAVis is particularly dedicated to assist both tutors and students…

Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

PubMed Central

Irani, Niloufer G; Grotewold, Erich

2005-01-01

Background Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS) cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. Results BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. Conclusion By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI)-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin. PMID:15907203

Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

PubMed

Lunprom, Siriporn; Pongcharoen, Pongsanat; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi; Akiyama, Koichi

2015-01-01

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

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

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

CpG methylation of a silent controlling element in the murine Avy allele is incomplete and unresponsive to methyl donor supplementation.

PubMed

Cropley, Jennifer E; Suter, Catherine M; Beckman, Kenneth B; Martin, David I K

2010-02-04

The viable yellow allele of agouti (A(vy)) is remarkable for its unstable and partially heritable epigenetic state, which produces wide variation in phenotypes of isogenic mice. In the A(vy) allele an inserted intracisternal A particle (IAP) acts as a controlling element which deregulates expression of agouti by transcription from the LTR of the IAP; the phenotypic state has been linked to CpG methylation of the LTR. Phenotypic variation between A(vy) mice indicates that the epigenetic state of the IAP is unstable in the germline. We have made a detailed examination of somatic methylation of the IAP using bisulphite allelic sequencing, and find that the promoter is incompletely methylated even when it is transcriptionally silent. In utero exposure to supplementary methyl donors, which alters the spectrum of A(vy) phenotypes, does not increase the density of CpG methylation in the silent LTR. Our findings suggest that, contrary to previous supposition, methyl donor supplementation acts through an indirect mechanism to silence A(vy). The incomplete cytosine methylation we observe at the somatically silent A(vy) allele may reflect its unstable germline state, and the influence of epigenetic modifications underlying CpG methylation.

CpG Methylation of a Silent Controlling Element in the Murine Avy Allele Is Incomplete and Unresponsive to Methyl Donor Supplementation

PubMed Central

Cropley, Jennifer E.; Suter, Catherine M.; Beckman, Kenneth B.; Martin, David I. K.

2010-01-01

Background The viable yellow allele of agouti (Avy) is remarkable for its unstable and partially heritable epigenetic state, which produces wide variation in phenotypes of isogenic mice. In the Avy allele an inserted intracisternal A particle (IAP) acts as a controlling element which deregulates expression of agouti by transcription from the LTR of the IAP; the phenotypic state has been linked to CpG methylation of the LTR. Phenotypic variation between Avy mice indicates that the epigenetic state of the IAP is unstable in the germline. Principal Findings We have made a detailed examination of somatic methylation of the IAP using bisulphite allelic sequencing, and find that the promoter is incompletely methylated even when it is transcriptionally silent. In utero exposure to supplementary methyl donors, which alters the spectrum of Avy phenotypes, does not increase the density of CpG methylation in the silent LTR. Conclusions Our findings suggest that, contrary to previous supposition, methyl donor supplementation acts through an indirect mechanism to silence Avy. The incomplete cytosine methylation we observe at the somatically silent Avy allele may reflect its unstable germline state, and the influence of epigenetic modifications underlying CpG methylation. PMID:20140227

Academic Visual Identity (AVI): An Act of Symbolic Leadership

ERIC Educational Resources Information Center

Masiki, Trent

2011-01-01

Extensive scholarship exists on both symbolic leadership and corporate visual identity (CVI), yet little scholarly attention has been focused on the intersection of these two bodies of knowledge. In the field of education, that intersection is known as academic visual identity (AVI). The purpose of this article is to demonstrate that AVI…

Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice.

PubMed

Wolff, G L; Kodell, R L; Moore, S R; Cooney, C A

1998-08-01

'Viable yellow' (Avy/a) mice are larger, obese, hyperinsulinemic, more susceptible to cancer, and, on average, shorter lived than their non-yellow siblings. They are epigenetic mosaics ranging from a yellow phenotype with maximum ectopic agouti overexpression, through a continuum of mottled agouti/yellow phenotypes with partial agouti overexpression, to a pseudoagouti phenotype with minimal ectopic expression. Pseudoagouti Avy/a mice are lean, healthy, and longer lived than their yellow siblings. Here we report that feeding pregnant black a/a dams methyl-supplemented diets alters epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling in the direction of the pseudoagouti phenotype. We also present confirmatory evidence that epigenetic phenotypes are maternally heritable. Thus Avy expression, already known to be modulated by imprinting, strain-specific modification, and maternal epigenetic inheritance, is also modulated by maternal diet. These observations suggest, at least in this special case, that maternal dietary supplementation may positively affect health and longevity of the offspring. Therefore, this experimental system should be useful for identifying maternal factors that modulate epigenetic mechanisms, especially DNA methylation, in developing embryos.

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.

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.

Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study

PubMed Central

Kinali, Maria; Arechavala-Gomeza, Virginia; Feng, Lucy; Cirak, Sebahattin; Hunt, David; Adkin, Carl; Guglieri, Michela; Ashton, Emma; Abbs, Stephen; Nihoyannopoulos, Petros; Garralda, Maria Elena; Rutherford, Mary; Mcculley, Caroline; Popplewell, Linda; Graham, Ian R; Dickson, George; Wood, Matthew JA; Wells, Dominic J; Wilton, Steve D; Kole, Ryszard; Straub, Volker; Bushby, Kate; Sewry, Caroline; Morgan, Jennifer E; Muntoni, Francesco

2009-01-01

Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen

Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E.

PubMed

Maurischat, Sven; Szabo, Istvan; Baumann, Beatrice; Malorny, Burkhard

2015-05-01

Salmonella enterica serovar Enteritidis is a major non-typhoid Salmonella serovar causing human salmonellosis mainly associated with the consumption of poultry and products thereof. To reduce infections in poultry, S. Enteritidis live vaccine strains AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE have been licensed and used in several countries worldwide. To definitively diagnose a S. Enteritidis contamination in vaccinated herds a reliable and fast method for the differentiation between vaccine and wildtype field isolates is required. In this study, we developed and validated real-time PCR (qPCR) assays to distinguish those variants genetically. Suitable target sequences were identified by whole genome sequencing (WGS) using the Illumina MiSeq system. SNP regions in kdpA and nhaA proved to be most useful for differentiation of AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE, respectively, from wildtype strains. For each vaccine strain one TaqMan-qPCR assay and one alternative approach using High Resolution Melting (HRM) analysis was designed. All 30 Salmovac SE and 7 AviPro SALMONELLA VAC E vaccine strain reisolates tested were correctly identified by both approaches (100% inclusivity). Furthermore, all 137 (TaqMan) and 97 (HRM) Salmonella non-vaccine and related Enterobacteriaceae strains tested were excluded (100% exclusivity). The analytical detection limits were determined to be approx. 10(2) genome copies/reaction for the TaqMan and 10(4) genome copies/reaction for the HRM approach. The real-time PCR assays proved to be a reliable and fast alternative to the cultural vaccine strain identification tests helping decision makers in control measurements to take action within a shorter period of time. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

PubMed

Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

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.

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

PubMed

Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

2008-12-01

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

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

2016-12-01

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3 (∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3 + but was not completely rescued by the expression of avt3 (∆1-270) . The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

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.

The AVI project: A bibliographical and archive inventory of landslides and floods in Italy

NASA Astrophysics Data System (ADS)

Guzzetti, Fausto; Cardinali, Mauro; Reichenbach, Paola

1994-07-01

The AVI project was commissioned by the Minister of Civil Protection to the National Group for Prevention of Hydrogeologic Hazards to complete an inventory of areas historically affected by landslides and floods in Italy. More than 300 people, divided into 15 research teams and two support groups, worked for one year on the project. Twenty-two journals were systematically searched for the period 1918 1990, 350,000 newspaper issues were screened, and 39,953 articles were collected. About 150 experts on mass movement and floods were interviewed and 1482 published and unpublished technical and scientific reports were reviewed. The results of the AVI project, in spite of the limitations, represent the most comprehensive archiving of mass movement and floods ever prepared in Italy. The type and quality of the information collected and the methodologies and techniques used to make the inventory are discussed. Possible applications and future developments are also presented.

Sensitivity and specificity of the Akena Visual Depression Inventory (AViDI-18) in Kampala (Uganda) and Cape Town (South Africa).

PubMed

Akena, Dickens; Joska, John; Stein, Dan J

2018-05-01

Visual scales may be particularly useful in screening for depression in patients with low literacy. However, few have been validated and none are in common use.AimModification and validation of a visual scale to screen for depression in low literacy settings. We assessed the validity, reliability and factor loading of a 28-item visual depression inventory using pictorial items depicting depression signs and symptoms. We validated a revised scale comprised of 18 items known as the Akena Visual Depression Inventory (AViDI-18) against a structured diagnostic interview (Mini-International Neuropsychiatric Inventory) in 343 patients in Kampala (Uganda) and Cape Town (South Africa). The 18 pictorial items had acceptable validity and reliability. The area under the curve (AUC) score of the AViDI-18 was 0.9. AUC scores were not significantly associated with sociodemographic variables. The AViDI-18 is a valid screen for depression in patients with low literacy.Declaration of interestNone.

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe.

PubMed

Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Morita, Tomotake; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2008-06-25

We have identified the Schizosaccharomyces pombe SPBC3E7.06c gene (fnx2(+)) from a homology search with the fnx1(+) gene involving in G(0) arrest upon nitrogen starvation. Green fluorescent protein-fused Fnx1p and Fnx2p localized exclusively to the vacuolar membrane. Uptake of histidine or isoleucine by S. pombe cells was inhibited by concanamycin A, a specific inhibitor of the vacuolar H(+)-ATPase. Amino acid uptake was also defective in the vacuolar ATPase mutant, suggesting that vacuolar compartmentalization is critical for amino acid uptake by whole cells. In both Deltafnx1 and Deltafnx2 mutant cells, uptake of lysine, isoleucine or asparagine was impaired. These results suggest that fnx1(+) and fnx2(+) are involved in vacuolar amino acid uptake in S. pombe.

A glycosylated recombinant human granulocyte colony stimulating factor produced in a novel protein production system (AVI-014) in healthy subjects: a first-in human, single dose, controlled study

PubMed Central

Varki, Roslyn; Pequignot, Ed; Leavitt, Mark C; Ferber, Andres; Kraft, Walter K

2009-01-01

Background AVI-014 is an egg white-derived, recombinant, human granulocyte colony-stimulating factor (G-CSF). This healthy volunteer study is the first human investigation of AVI-014. Methods 24 male and female subjects received a single subcutaneous injection of AVI-014 at 4 or 8 mcg/kg. 16 control subjects received 4 or 8 mcg/kg of filgrastim (Neupogen, Amgen) in a partially blinded, parallel fashion. Results The Geometric Mean Ratio (GMR) (90% CI) of 4 mcg/kg AVI-014/filgrastim AUC(0–72 hr) was 1.00 (0.76, 1.31) and Cmax was 0.86 (0.66, 1.13). At the 8 mcg/kg dose, the AUC(0–72) GMR was 0.89 (0.69, 1.14) and Cmax was 0.76 (0.58, 0.98). A priori pharmacokinetic bioequivalence was defined as the 90% CI of the GMR bounded by 0.8–1.25. Both the white blood cell and absolute neutrophil count area under the % increase curve AUC(0–9 days) and Cmax (maximal % increase from baseline)GMR at 4 and 8 mcg/kg fell within the 0.5–2.0 a priori bound set for pharmacodynamic bioequivalence. The CD 34+ % increase curve AUC(0–9 days) and Cmax GMR for both doses was ~1, but 90% confidence intervals were large due to inherent variance, and this measure did not meet pharmacodynamic bioequivalence. AVI-014 demonstrated a side effect profile similar to that of filgrastim. Conclusion AVI-014 has safety, pharmacokinetic, and pharmacodynamic properties comparable to filgrastim at an equal dose in healthy volunteers. These findings support further investigation in AVI-014. PMID:19175929

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

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

Soy protein isolate reduces hepatosteatosis in yellow Avy/a mice without altering coat color phenotype

USDA-ARS?s Scientific Manuscript database

Agouti (Avy/a) mice fed an AIN-93G diet containing the soy isoflavone genistein (GEN) prior to and during pregnancy were reported to shift coat color and body composition phenotypes from obese-yellow towards lean pseudoagouti, suggesting epigenetic programming. Human consumption of purified GEN is r...

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields.

PubMed

Marin Viegas, Vanesa Soledad; Ocampo, Carolina Gabriela; Petruccelli, Silvana

2017-05-04

Delivery of recombinant proteins to vegetative tissue vacuoles was considered inconvenient since this compartment was expected to be hydrolytic; nevertheless there is growing evidence that certain foreign proteins accumulate at high yields in vacuoles. For example avidin, cellulolytic enzymes, endolysin, and transglutaminases were produced at high yields when were sorted to leaf central vacuole avoiding the detrimental effect of these proteins on plant growth. Also, several secretory mammalian proteins such as collagen, α1-proteinase inhibitor, complement-5a, interleukin-6 and immunoglobulins accumulated at higher yields in leaf vacuoles than in the apoplast or cytosol. To reach this final destination, fusions to sequence specific vacuolar sorting signals (ssVSS) typical of proteases or proteinase inhibitors and/or Ct-VSS representative of storage proteins or plant lectins were used and both types of motifs were capable to increase accumulation. Importantly, the type of VSSs or position, either the N or C-terminus, did not alter protein stability, levels or pos-translational modifications. Vacuolar sorted glycoproteins had different type of oligosaccharides indicating that foreign proteins reached the vacuole by 2 different pathways: direct transport from the ER, bypassing the Golgi (high mannose oligosaccharides decorated proteins) or trafficking through the Golgi (Complex oligosaccharide containing proteins). In addition, some glycoproteins lacked of paucimannosidic oligosaccharides suggesting that vacuolar trimming of glycans did not occur. Enhanced accumulation of foreign proteins fused to VSS occurred in several plant species such as tobacco, Nicotiana benthamiana, sugarcane, tomato and in carrot and the obtained results were influenced by plant physiological state. Ten different foreign proteins fused to vacuolar sorting accumulated at higher levels than their apoplastic or cytosolic counterparts. For proteins with cytotoxic effects vacuolar sorted forms

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

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.

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.

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.

Influence of Background Genome on Enzymatic Characteristics of Yellow (Ay/-, Avy/-) Mice

PubMed Central

Wolff, George L.; Pitot, Henry C.

1973-01-01

Identification of the fundamental polypeptide difference between yellow (Ay/-, Avy/-) and non-yellow mice is important for biomedical research because of the influence of the yellow genotype on normal and neoplastic growth and obesity. The complexity of the "yellow mouse syndrome" makes attainment of this objective dependent on the separation of those pleiotropic enzyme differences which are secondary, and depend on the background genome, from those which are primary, and depend primarily on the agouti locus genotype.—Four of nine hepatic enzyme activities assayed simultaneously differed between eight-week-old yellow (Ay/-, Avy/-) and non-yellow (A/-, a/a) male inbred and F1 hybrid mice. Among these four, only cytoplasmic malic enzyme activity was elevated in all yellow mice, as compared with the non-yellow sibs, regardless of background genome. Glucokinase, serine dehydratase, and tyrosine α-ketoglutarate transaminase activities were also changed in yellow mice, but these alterations depended on the background genome.—The ratio of malic enzyme activity to citrate-cleavage enzyme activity, possibly related to the altered fat metabolism of yellow mice, was influenced by background genome as well as by the yellow genotype.——Significant deviations of enzyme activities from mid-parent values among F1 hybrids were associated with particular background genomes; the number of such deviations was larger among yellow mice than among non-yellows and this difference was greater among C3H F1 hybrids than among C57BL/6 F1 hybrids. PMID:4405752

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

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.

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

Paired Helical Filaments of Inclusion-Body Myositis Muscle Contain RNA and Survival Motor Neuron Protein

PubMed Central

Broccolini, Aldobrando; Engel, W. King; Alvarez, Renate B.; Askanas, Valerie

2000-01-01

Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older persons. Pathologically, the muscle biopsy manifests various degrees of inflammation and specific vacuolar degeneration of muscle fibers characterized by paired helical filaments (PHFs) composed of phosphorylated tau. IBM vacuolated fibers also contain accumulations of several other Alzheimer-characteristic proteins. Molecular mechanisms leading to formation of the PHFs and accumulations of proteins in IBM muscle are not known. We report that the abnormal muscle fibers of IBM contained (i) acridine-orange-positive RNA inclusions that colocalized with the immunoreactivity of phosphorylated tau and (ii) survival motor neuron protein immunoreactive inclusions, which by immuno-electron microscopy were confined to paired helical filaments. This study demonstrates two novel components of the IBM paired helical filaments, which may lead to better understanding of their pathogenesis. PMID:10751338

Paired helical filaments of inclusion-body myositis muscle contain RNA and survival motor neuron protein.

PubMed

Broccolini, A; Engel, W K; Alvarez, R B; Askanas, V

2000-04-01

Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older persons. Pathologically, the muscle biopsy manifests various degrees of inflammation and specific vacuolar degeneration of muscle fibers characterized by paired helical filaments (PHFs) composed of phosphorylated tau. IBM vacuolated fibers also contain accumulations of several other Alzheimer-characteristic proteins. Molecular mechanisms leading to formation of the PHFs and accumulations of proteins in IBM muscle are not known. We report that the abnormal muscle fibers of IBM contained (i) acridine-orange-positive RNA inclusions that colocalized with the immunoreactivity of phosphorylated tau and (ii) survival motor neuron protein immunoreactive inclusions, which by immuno-electron microscopy were confined to paired helical filaments. This study demonstrates two novel components of the IBM paired helical filaments, which may lead to better understanding of their pathogenesis.

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

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.

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.

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 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.

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

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.

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.

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.

Marriage and Family Therapists Working with Family Violence: Strained Bedfellows or Compatible Partners?: A Commentary on Avis, Kaufman, and Bograd.

ERIC Educational Resources Information Center

Meth, Richard L.

1992-01-01

Responds to previous articles by Avis, Kaufman, and Bograd on role of marital and family therapists in dealing with family violence among clients. Comments on presentation style of earlier articles and then discusses points of agreement and disagreement with each of the three authors. Concludes by urging therapists to learn more so they can…

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

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

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

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

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.

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.

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

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.

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.

Vacuolar invertase gene silencing in potato decreasing the frequency of sugar-end defects

USDA-ARS?s Scientific Manuscript database

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one e...

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

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

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

[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.

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

PubMed Central

Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

2015-01-01

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

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

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

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.

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

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.

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.

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.

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

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.

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.

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

Exposure to Soy Protein Isolate From Conception Fails to Induce Epigenetic Changes in Viable Yellow Agouti (Avy/a) Mice, But Partially Blocks Hepatosteatosis and Altered Body Composition in Mice and Rats

USDA-ARS?s Scientific Manuscript database

Both beneficial and adverse health effects have been attributed to soy food consumption. Epigenetic programming through hypermethlylation of CpG sites on promoter regions may be a potential mechanism. Virgin a/a female and Avy/a male mice were fed AIN-93G diets made with either casein or soy protein...

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...

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.

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

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

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.

Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects

USDA-ARS?s Scientific Manuscript database

Sugar-end defect is a tuber quality disorder that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form da...

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.

Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions.

PubMed

Askanas, V; Engel, W K; Alvarez, R B; McFerrin, J; Broccolini, A

2000-07-01

Alpha-synuclein (alpha-syn) is an important component of neuronal and glial inclusions in brains of patients with several neurodegenerative disorders. Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older patients. Its muscle phenotype shows several similarities with Alzheimer disease brain. A distinct feature of s-IBM pathology is specific vacuolar degeneration of muscle fibers characterized by intracellular amyloid inclusions formed by both amyloid-beta (Abeta) and paired-helical filaments composed of phosphorylated tau. We immunostained alpha-syn in muscle biopsies of s-IBM, disease-control, and normal patients. Approximately 60% of Abeta-positive vacuolated muscle fibers (VMF) contained well-defined inclusions immunoreactive with antibodies against alpha-syn. In those fibers. alpha-syn co-localized with Abeta, both by light microscopy, and ultrastructurally. Paired-helical filaments did not contain alpha-syn immunoreactivity. In all muscle biopsies, alpha-syn was strongly immunoreactive at the postsynaptic region of the neuromuscular junctions. alpha-syn immunoreactivity also occurred diffusely in regenerating and necrotic muscle fibers. In cultured human muscle fibers, alpha-syn and its mRNA were expressed by immunocytochemistry, immunoblots, and Northern blots. Our study provides the first demonstration that alpha-syn participates in normal and pathologic processes of human muscle. Therefore. its function is not exclusive to the brain and neurodegenerative diseases.

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.

Dietary protection against free radicals: a case for multiple testing to establish structure-activity relationships for antioxidant potential of anthocyanic plant species.

PubMed

Philpott, Martin; Lim, Chiara Cheng; Ferguson, Lynnette R

2009-03-01

DNA damage by reactive species is associated with susceptibility to chronic human degenerative disorders. Anthocyanins are naturally occurring antioxidants, that may prevent or reverse such damage. There is considerable interest in anthocyanic food plants as good dietary sources, with the potential for reducing susceptibility to chronic disease. While structure-activity relationships have provided guidelines on molecular structure in relation to free hydroxyl-radical scavenging, this may not cover the situation in food plants where the anthocyanins are part of a complex mixture, and may be part of complex structures, including anthocyanic vacuolar inclusions (AVIs). Additionally, new analytical methods have revealed new structures in previously-studied materials. We have compared the antioxidant activities of extracts from six anthocyanin-rich edible plants (red cabbage, red lettuce, blueberries, pansies, purple sweetpotato skin, purple sweetpotato flesh and Maori potato flesh) using three chemical assays (DPPH, TRAP and ORAC), and the in vitro Comet assay. Extracts from the flowering plant, lisianthus, were used for comparison. The extracts showed differential effects in the chemical assays, suggesting that closely related structures have different affinities to scavenge different reactive species. Integration of anthocyanins to an AVI led to more sustained radical scavenging activity as compared with the free anthocyanin. All but the red lettuce extract could reduce endogenous DNA damage in HT-29 colon cancer cells. However, while extracts from purple sweetpotato skin and flesh, Maori potato and pansies, protected cells against subsequent challenge by hydrogen peroxide at 0 degrees C, red cabbage extracts were pro-oxidant, while other extracts had no effect. When the peroxide challenge was at 37 degrees C, all of the extracts appeared pro-oxidant. Maori potato extract, consistently the weakest antioxidant in all the chemical assays, was more effective in the

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 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.

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.

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.

The Inclusive Classroom: How Inclusive Is Inclusion?

ERIC Educational Resources Information Center

Reid, Claudette M.

2010-01-01

This paper presents the position that inclusion is limited; inclusion does not go far enough. The inclusive classroom has been assessed to be of benefit both to the teacher and student. There are, however, limits set on inclusion. In most classrooms only children with learning disability are included omitting those with severe disabilities,…

Vacuolar ATPase in Phagosome-Lysosome Fusion

PubMed Central

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-01-01

The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

Vacuolar ATPase in phagosome-lysosome fusion.

PubMed

Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

2015-05-29

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: a fluorescence microscopy analysis.

PubMed

Tattini, Massimiliano; Di Ferdinando, Martina; Brunetti, Cecilia; Goti, Andrea; Pollastri, Susanna; Bellasio, Chandra; Giordano, Cristiana; Fini, Alessio; Agati, Giovanni

2014-11-01

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance was estimated using their specific UV- and fluorescence spectral features. Using a combination of UV-induced fluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-amino-ethylester, in wide field or confocal laser scanning microscopy, we were able to visualize the distribution of esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributed in the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeed shows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previously reported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a different vacuolar distribution: esculetin largely predominates in the first 15 μm from the adaxial epidermis. This leads to hypothesize for esculetin and esculin different transport mechanisms from the endoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our study open to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole using different fluorescence signatures of coumarin aglycones and coumarin glycosides. Copyright © 2014 Elsevier B.V. All rights reserved.

vph6 mutants of Saccharomyces cerevisiae require calcineurin for growth and are defective in vacuolar H(+)-ATPase assembly.

PubMed

Hemenway, C S; Dolinski, K; Cardenas, M E; Hiller, M A; Jones, E W; Heitman, J

1995-11-01

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.

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.

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.

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

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.

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.

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.

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.

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.

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.

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

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.

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

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

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.

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.

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.

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.

Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

PubMed

Zheng, Desen; Burr, Thomas J

2016-02-01

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

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.

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...

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.

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

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.

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.

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

An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism Streptomyces viridochromogenes Tü57

PubMed Central

Weitnauer, Gabriele; Gaisser, Sibylle; Trefzer, Axel; Stockert, Sigrid; Westrich, Lucy; Quiros, Luis M.; Mendez, Carmen; Salas, Jose A.; Bechthold, Andreas

2001-01-01

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA. PMID:11181344

An ATP-binding cassette transporter and two rRNA methyltransferases are involved in resistance to avilamycin in the producer organism Streptomyces viridochromogenes Tü57.

PubMed

Weitnauer, G; Gaisser, S; Trefzer, A; Stockert, S; Westrich, L; Quiros, L M; Mendez, C; Salas, J A; Bechthold, A

2001-03-01

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

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

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

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.

An Sfp-type PPTase and associated polyketide and nonribosomal peptide synthases in Agrobacterium vitis are essential for induction of tobacco hypersensitive response and grape necrosis.

PubMed

Zheng, Desen; Burr, Thomas J

2013-07-01

An Sfp-type phosphopantetheinyl transferase (PPTase) encoding gene F-avi5813 in Agrobacterium vitis F2/5 was found to be required for the induction of a tobacco hypersensitive response (HR) and grape necrosis. Sfp-type PPTases are post-translation modification enzymes that activate acyl-carry protein (ACP) domains in polyketide synthases (PKS) and peptidyl-carrier protein (PCP) domains of nonribosomal peptide synthases (NRPS). Mutagenesis of PKS and NRPS genes in A. vitis led to the identification of a PKS gene (F-avi4330) and NRPS gene (F-avi3342) that are both required for HR and necrosis. The gene immediately downstream of F-avi4330 (F-avi4329) encoding a predicted aminotransferase was also found to be required for HR and necrosis. Regulation of F-avi4330 and F-avi3342 by quorum-sensing genes avhR, aviR, and avsR and by a lysR-type regulator, lhnR, was investigated. It was determined that F-avi4330 expression is positively regulated by avhR, aviR, and lhnR and negatively regulated by avsR. F-avi3342 was found to be positively regulated by avhR, aviR, and avsR and negatively regulated by lhnR. Our results suggest that a putative hybrid peptide-polyketide metabolite synthesized by F-avi4330 and F-avi3342 is associated with induction of tobacco HR and grape necrosis. This is the first report that demonstrates that NRPS and PKS play essential roles in conferring the unique ability of A. vitis to elicit a non-host-specific HR and host-specific necrosis.

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.

Inclusive indoor play: an approach to developing inclusive design guidelines.

PubMed

Mullick, Abir

2013-01-01

The purpose of the Inclusive Indoor Play study was to learn about indoor play and develop design guidelines to inform design of inclusive playthings. Children with and without disabilities, parents, teachers, therapists, daycare owners and designers. Focus group interviews; Children's drawings; and Indoor play simulation. The major findings suggest that: 1) play should encourage a child's creativity and develop imagination, 2) inclusive play concept must be employed to design playthings for children with wide age group, 3) inclusive designs improve usability, broaden market appeal, and increase user base, and 4) customizable playthings help children with and without disabilities personalize play situations. Three play principles provide new directions to designing inclusive playthings: 1) offer many play opportunities, 2) provide many modes of play, and 3) include many levels of play challenges. Inclusive Design Guidelines were developed from the findings of three studies: Focus group interviews, Children's drawings, and Play simulation. The guidelines served as useful tools for inclusive design and they were employed to design of six indoor playthings. The playthings were instrumental in promoting social inclusion and they met the criteria of the Inclusive Indoor Play project.

Inclusive health.

PubMed

Maclachlan, Malcolm; Khasnabis, Chapal; Mannan, Hasheem

2012-01-01

We propose the concept of Inclusive Health to encapsulate the Health for All ethos; to build on the rights-based approach to health; to promote the idea of inclusion as a verb, where a more proactive approach to addressing distinctive and different barriers to inclusion is needed; and to recognise that new initiatives in human resources for health can offer exciting and innovative ways of healthcare delivery. While Inclusive Education has become a widely recognised and accepted concept, Health for All is still contested, and new thinking is required to develop its agenda in line with contemporary developments. Inclusive Health refers both to who gets health care and to who provides it; and its ethos resonates strongly with Jefferson's assertion that 'there is nothing more unequal, than the equal treatment of unequal people'. We situate the timeliness of the Inclusive Health concept with reference to recent developments in the recognition of the rights of people with disability, in the new guidelines for community-based rehabilitation and in the World Report on Disability. These developments offer a more inclusive approach to health and, more broadly, its inter-connected aspects of wellbeing. A concept which more proactively integrates United Nations conventions that recognise the importance of difference - disability, ethnicity, gender, children - could be of benefit for global healthcare policy and practice. © 2011 Blackwell Publishing Ltd.

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.

Inclusion.

ERIC Educational Resources Information Center

Nathanson, Jeanne H., Ed.

1992-01-01

This theme journal issue focuses on current activities of the Office of Special Education and Rehabilitative Services which stress inclusion of students with disabilities in the mainstream. It begins with a message from the Assistant Secretary, Robert R. Davila which examines the full meaning of an "inclusive" education. Next, Barbara…

Questioning Secondary Inclusive Education: Are Inclusive Classrooms Always Best for Students?

ERIC Educational Resources Information Center

Tkachyk, Ruth Elizabeth

2013-01-01

Educating students with special needs in inclusive settings has become a priority for westernized governments as they strive to create more inclusive societies. While recognizing the societal benefits of inclusion, teachers and parents question whether or not implementation of full inclusion will come at the expense of learners' individual…

Limits to Inclusion

ERIC Educational Resources Information Center

Hansen, Janne Hedegaard

2012-01-01

In this article, I will argue that a theoretical identification of the limit to inclusion is needed in the conceptual identification of inclusion. On the one hand, inclusion is formulated as a vision that is, in principle, limitless. On the other hand, there seems to be an agreement that inclusion has a limit in the pedagogical practice. However,…

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.

Safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single-ascending-dose studies.

PubMed

Heald, Alison E; Iversen, Patrick L; Saoud, Jay B; Sazani, Peter; Charleston, Jay S; Axtelle, Tim; Wong, Michael; Smith, William B; Vutikullird, Apinya; Kaye, Edward

2014-11-01

Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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.

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.

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.

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.

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

Inclusive Education in Italy: Description and Reflections on Full Inclusion

ERIC Educational Resources Information Center

Anastasiou, Dimitris; Kauffman, James M.; Di Nuovo, Santo

2015-01-01

Inclusion of students with disabilities when appropriate is an important goal of special education for students with special needs. Full inclusion, meaning no education for any child in a separate setting, is held to be desirable by some, and Italy is likely the nation with an education system most closely approximating full inclusion on the…

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.

The role of repeating optimization of atrioventricular interval during interim and long-term follow-up after cardiac resynchronization therapy.

PubMed

Zhang, Qing; Fung, Jeffrey Wing-Hong; Chan, Yat-Sun; Chan, Hamish Chi-Kin; Lin, Hong; Chan, Skiva; Yu, Cheuk-Man

2008-02-29

Cardiac resynchronization therapy (CRT) is an effective therapy for heart failure patients with electromechanical delay. Optimization of atrioventricular interval (AVI) is a cardinal component for the benefits. However, it is unknown if the AVI needs to be re-optimized during long-term follow-up. Thirty-one patients (66+/-11 years, 20 males) with sinus rhythm who received CRT underwent serial optimization of AVI at day 1, 3-month and during long-term follow-up by pulse Doppler echocardiography (PDE). At long-term follow-up, the optimal AVI and cardiac output (CO) estimated by non-invasive impedance cardiography (ICG) were compared with those by PDE. The follow-up was 16+/-11 months. There was no significant difference in the mean optimal AVI when compared between any 2 time points among day 1 (99+/-30 ms), 3-month (97+/-28 ms) and long-term follow-up (94+/-28 ms). However, in individual patient, the optimal AVI remained unchanged only in 14 patients (44%), and was shortened in 12 (38%) and lengthened in 6 patients (18%). During long-term follow-up, although the mean optimal AVIs obtained by PDE or ICG (94+/-28 vs. 92+/-29 ms) were not different, a discrepancy was found in 14 patients (45%). For the same AVI, the CO measured by ICG was systematically higher than that by PDE (3.5+/-0.8 Vs. 2.7+/-0.6 L/min, p<0.001). Optimization of AVI after CRT appears necessary during follow-up as it was readjusted in 55% of patients. Although AVI optimization by ICG was feasible, further studies are needed to confirm its role in optimizing AVI after CRT.

Phenotypic variability within the inclusion body spectrum of basophilic inclusion body disease and neuronal intermediate filament inclusion disease in frontotemporal lobar degenerations with FUS-positive inclusions.

PubMed

Gelpi, Ellen; Lladó, Albert; Clarimón, Jordi; Rey, Maria Jesús; Rivera, Rosa Maria; Ezquerra, Mario; Antonell, Anna; Navarro-Otano, Judith; Ribalta, Teresa; Piñol-Ripoll, Gerard; Pérez, Anna; Valldeoriola, Francesc; Ferrer, Isidre

2012-09-01

Basophilic inclusion body disease and neuronal intermediate filament inclusion disease (NIFID) are rare diseases included among frontotemporal lobar degenerations with FUS-positive inclusions (FTLD-FUS). We report clinical and pathologic features of 2 new patients and reevaluate neuropathologic characteristics of 2 previously described cases, including an early-onset case of basophilic inclusion body disease (aged 38 years) with a 5-year disease course and abundant FUS-positive inclusion bodies and 3 NIFID cases. One NIFID case (aged 37 years) presented with early-onset psychiatric disturbances and rapidly progressive cognitive decline. Two NIFID cases had later onset (aged 64 years and 70 years) and complex neurologic deficits. Postmortem neuropathologic studies in late-onset NIFID cases disclosed α-internexin-positive "hyaline conglomerate"-type inclusions that were positive with 1 commercial anti-FUS antibody directed to residues 200 and 250, but these were negative to amino acids 90 and 220 of human FUS. Early-onset NIFID had similar inclusions that were positive with both commercial anti-FUS antibodies. Genetic testing performed on all cases revealed no FUS gene mutations. These findings indicate that phenotypic variability in NIFID, including clinical manifestations and particular neuropathologic findings, may be related to the age at onset and individual differences in the evolution of lesions.

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57.

PubMed Central

Gaisser, S; Trefzer, A; Stockert, S; Kirschning, A; Bechthold, A

1997-01-01

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins. PMID:9335272

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion.

PubMed

Karim, Mahmoud Abdul; Brett, Christopher Leonard

2018-02-01

Loss-of-function mutations in human endosomal Na + (K + )/H + exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson syndrome, autism, and attention deficit and hyperactivity disorder. These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not the vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function. © 2018 Karim and Brett. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

The vacuolar protein sorting genes in insects: A comparative genome view.

PubMed

Li, Zhaofei; Blissard, Gary

2015-07-01

In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inclusive Education: Identifying Teachers' Perceived Stressors in Inclusive Classrooms

ERIC Educational Resources Information Center

Brackenreed, Darlene

2008-01-01

This research replicates the study conducted by Forlin (2001) in Churchlands, Western Australia. Forlin's Inclusive Education Teacher Stress and Coping Questionnaire was adapted from the original questionnaire to more accurately reflect the language and practice of inclusion in Ontario (Frost & Brackenreed, 2004). The purpose of this study was…

Beyond Physical Inclusion: Teaching Skills in the Community to Enhance Social Inclusion

ERIC Educational Resources Information Center

Hall, Carmen L.

2017-01-01

Along with the deinstitutionalization movement, supports for persons with Intellectual Disabilities (ID) have shifted to promotion of person-centered supports inclusive in the community. Although successes have occurred regarding physical inclusion, skill building and social inclusion have not fared as well for those with more significant…

Inclusion by Design: Engineering Inclusive Practices in Secondary Schools

ERIC Educational Resources Information Center

Dukes, Charles; Lamar-Dukes, Pamela

2009-01-01

In order to help teachers understand the importance of intentional design for inclusive education, this article describes the design process an engineer might use when designing a new project. If teachers learn to think like engineers, it is possible for them to design inclusive education. This conceptual design can then be combined with…

The crack-inclusion interaction problem

NASA Technical Reports Server (NTRS)

Liu, X.-H.; Erdogan, F.

1986-01-01

The general plane elastostatic problem of interaction between a crack and an inclusion is considered. The Green's functions for a pair of dislocations and a pair of concentrated body forces are used to generate the crack and the inclusion. Integral equations are obtained for a line crack and an elastic line inclusion having an arbitrary relative orientation and size. The nature of stress singularity around the end points of rigid and elastic inclusions is described and three special cases of this intersection problem are studied. The problem is solved for an arbitrary uniform stress state away from the crack-inclusion region. The nonintersecting crack-inclusion problem is considered for various relative size, orientation, and stiffness parameters, and the stress intensity factors at the ends of the inclusion and the crack are calculated. For the crack-inclusion intersection case, special stress intensity factors are defined and are calculated for various values of the parameters defining the relative size and orientation of the crack and the inclusion and the stiffness of the inclusion.

The crack-inclusion interaction problem

NASA Technical Reports Server (NTRS)

Xue-Hui, L.; Erdogan, F.

1984-01-01

The general plane elastostatic problem of interaction between a crack and an inclusion is considered. The Green's functions for a pair of dislocations and a pair of concentrated body forces are used to generate the crack and the inclusion. Integral equations are obtained for a line crack and an elastic line inclusion having an arbitrary relative orientation and size. The nature of stress singularity around the end points of rigid and elastic inclusions is described and three special cases of this intersection problem are studied. The problem is solved for an arbitrary uniform stress state away from the crack-inclusion region. The nonintersecting crack-inclusion problem is considered for various relative size, orientation, and stiffness parameters, and the stress intensity factors at the ends of the inclusion and the crack are calculated. For the crack-inclusion intersection case, special stress intensity factors are defined and are calculated for various values of the parameters defining the relative size and orientation of the crack and the inclusion and the stiffness of the inclusion.

Genotype-Specific Regulation of Oral Innate Immunity by T2R38 Taste Receptor

PubMed Central

Gil, Sucheol; Coldwell, Susan; Drury, Jeanie L.; Arroyo, Fabiola; Phi, Tran; Saadat, Sanaz; Kwong, Danny; Chung, Whasun Oh

2015-01-01

The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group. PMID

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

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

Milgrom, Elena M.; Milgrom, Yakov M., E-mail: milgromy@upstate.edu

2012-06-29

Highlights: Black-Right-Pointing-Pointer MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Black-Right-Pointing-Pointer V-ATPase activity saturation with MgATP is not sufficient for complete protection. Black-Right-Pointing-Pointer The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibitionmore » by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K{sub m} values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.« less

The Value of Inclusion

ERIC Educational Resources Information Center

Felder, Franziska

2018-01-01

In recent years inclusion has become one of the most dominant values and objectives in education. However, there is still considerable disagreement concerning the theoretical concept of inclusion and its normative implications. This article suggests an understanding of inclusion that first differentiates analytically between societal and communal…

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling*

PubMed Central

Lang, Michael J.; Martinez-Marquez, Jorge Y.; Prosser, Derek C.; Ganser, Laura R.; Buelto, Destiney; Wendland, Beverly; Duncan, Mara C.

2014-01-01

Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion. In the yeast Saccharomyces cerevisiae, glucose starvation causes an acute depletion of intracellular energy that recovers during continued glucose starvation. However, how the cell replenishes energy during the early phase of glucose starvation is unknown. In this study, we investigated the role of pathways that deliver proteins and lipids to the vacuole during glucose starvation. We report that in response to glucose starvation, plasma membrane proteins are directed to the vacuole through reduced recycling at the endosomes. Furthermore, we found that vacuolar hydrolysis inhibits macroautophagy in a target of rapamycin complex 1-dependent manner. Accordingly, we found that endocytosis and hydrolysis are required for survival in glucose starvation, whereas macroautophagy is dispensable. Together, these results suggest that hydrolysis of components delivered to the vacuole independent of autophagy is the cell survival mechanism used by S. cerevisiae in response to glucose starvation. PMID:24753258

SCF(TIR1/AFB)-auxin signalling regulates PIN vacuolar trafficking and auxin fluxes during root gravitropism.

PubMed

Baster, Paweł; Robert, Stéphanie; Kleine-Vehn, Jürgen; Vanneste, Steffen; Kania, Urszula; Grunewald, Wim; De Rybel, Bert; Beeckman, Tom; Friml, Jiří

2013-01-23

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-box(TIR1/AFB) (SCF(TIR1/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.

Evolutionary Divergence of Plant Borate Exporters and Critical Amino Acid Residues for the Polar Localization and Boron-Dependent Vacuolar Sorting of AtBOR1.

PubMed

Wakuta, Shinji; Mineta, Katsuhiko; Amano, Taro; Toyoda, Atsushi; Fujiwara, Toru; Naito, Satoshi; Takano, Junpei

2015-05-01

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A pivotal role of vacuolar H(+)-ATPase in regulation of lipid production in Phaeodactylum tricornutum.

PubMed

Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

2016-08-08

Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H(+)-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1.

Inclusion as Professional Development

ERIC Educational Resources Information Center

Stanovich, Paula J.; Jordan, Anne

2004-01-01

The inclusion of students with disabilities in general education classrooms has become the preferred model of service delivery in many educational jurisdictions. The benefits of an inclusive model for students are becoming clear as the research evidence begins to accumulate. However, the authors argue, inclusion may well offer benefits for…

Inclusion in Middle Tennessee

ERIC Educational Resources Information Center

Salter, Derrick; Ashley, Mandi; Hayes, Brandalyn

2013-01-01

The overall purpose of this study was to provide school districts within Tennessee with more research about how weekly hours of inclusion impact student achievement. Specifically, researchers examined which models of inclusion were in use in two school districts in Tennessee, administrators' and teachers' perceptions of inclusion, and whether or…

Inclusive Education in Bangladesh

ERIC Educational Resources Information Center

Ahsan, Mohammad Tariq; Burnip, Lindsay

2007-01-01

This article reports on inclusive education in Bangladesh for children with special needs. Bangladesh is not behind other developed countries in enacting laws and declarations in favour of inclusive education, but a lack of resources is the main barrier in implementing inclusive education. Special education and integrated education models exist in…

Footstep towards Inclusive Education

ERIC Educational Resources Information Center

Abbas, Faiza; Zafar, Aneeka; Naz, Tayyaba

2016-01-01

Inclusive education is a rising trend in the world. The first step towards inclusive education is providing the awareness to the general education teachers. This study focused to investigate the general education teachers of primary and secondary level awareness about the special education and inclusive education. This study is descriptive method…

More Policies, Greater Inclusion? Exploring the Contradictions of New Labour Inclusive Education Policy

ERIC Educational Resources Information Center

Roulstone, Alan; Prideaux, Simon

2008-01-01

The era of New Labour government has witnessed unprecedented growth in inclusive education policies. There is, however, limited evidence that policies have increased disabled children's inclusion. This article explores reasons for this contradiction. Drawing on sociological insights, it is argued that New Labour policies on inclusive education…

Linguistic Diversity and Social Inclusion

ERIC Educational Resources Information Center

Piller, Ingrid; Takahashi, Kimie

2011-01-01

This introduction provides the framework for the special issue by describing the social inclusion agenda of neoliberal market democracies. While the social inclusion agenda has been widely adopted, social inclusion policies are often blind to the ways in which language proficiency and language ideologies mediate social inclusion in linguistically…

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.

Inclusion in the East: Chinese Students' Attitudes towards Inclusive Education

ERIC Educational Resources Information Center

Malinen, Olli-Pekka; Savolainen, Hannu

2008-01-01

A sample of 523 Chinese university students was given a questionnaire on their attitudes towards the inclusion of children with disabilities into regular classrooms. Factor analysis, analysis of variance, t-test and correlations were used to assess the respondents' general attitude towards inclusion, the factor structure of the attitudes, the…

Homogenisation of sulphide inclusions within diamonds: A new approach to diamond inclusion geochemistry

NASA Astrophysics Data System (ADS)

McDonald, Iain; Hughes, Hannah S. R.; Butler, Ian B.; Harris, Jeffrey W.; Muir, Duncan

2017-11-01

Base metal sulphide (BMS) inclusions in diamonds provide a unique insight into the chalcophile and highly siderophile element composition of the mantle. Entombed within their diamond hosts, these provide a more robust (closed system) sample, from which to determine the trace element, Re-Os and S-isotopic compositions of the mantle than mantle xenoliths or orogenic peridotites, as they are shielded from alteration during ascent to the Earth's crust and subsequent surface weathering. However, at temperatures below 1100 °C some BMS inclusions undergo subsolidus re-equilibration from an original monosulphide solid solution (Mss) and this causes fractionation of the major and trace elements within the inclusions. Thus to study the subjects noted above, current techniques require the entire BMS inclusion to be extracted for analyses. Unfortunately, 'flaking' of inclusions during break-out is a frequent occurrence and hence the risk of accidentally under-sampling a portion of the BMS inclusion is inherent in current practices. This loss may have significant implications for Re-Os isotope analyses where incomplete sampling of a Re-rich phase, such as chalcopyrite that typically occurs at the outer margins of BMS inclusions, may induce significant bias in the Re-Os and 187Os/188Os measurements and resulting model and isochron ages. We have developed a method for the homogenisation of BMS inclusions in diamond prior to their break-out from the host stone. Diamonds are heated to 1100 °C and then quenched to chemically homogenise any sulphide inclusions for both major and trace elements. Using X-ray Computed Microtomography (μCT) we determine the shape and spatial setting of multiple inclusions within a host stone and crucially show that the volume of a BMS inclusion is the same both before and after homogenisation. We show that the homogenisation process significantly reduces the inherent variability of in situ analysis when compared with unhomogenised BMS, thereby

Measuring the Quality of Inclusive Practices: Findings from the Inclusive Classroom Profile Pilot

ERIC Educational Resources Information Center

Soukakou, Elena P.; Winton, Pam J.; West, Tracey A.; Sideris, John H.; Rucker, Lia M.

2014-01-01

The purpose of this study was to test the reliability and validity of the Inclusive Classroom Profile (ICP), an observation measure designed to assess the quality of classroom practices in inclusive preschool programs. The measure was field tested in 51 inclusive classrooms. Results confirmed and extended previous research findings, providing…

Limitations of inclusive fitness.

PubMed

Allen, Benjamin; Nowak, Martin A; Wilson, Edward O

2013-12-10

Until recently, inclusive fitness has been widely accepted as a general method to explain the evolution of social behavior. Affirming and expanding earlier criticism, we demonstrate that inclusive fitness is instead a limited concept, which exists only for a small subset of evolutionary processes. Inclusive fitness assumes that personal fitness is the sum of additive components caused by individual actions. This assumption does not hold for the majority of evolutionary processes or scenarios. To sidestep this limitation, inclusive fitness theorists have proposed a method using linear regression. On the basis of this method, it is claimed that inclusive fitness theory (i) predicts the direction of allele frequency changes, (ii) reveals the reasons for these changes, (iii) is as general as natural selection, and (iv) provides a universal design principle for evolution. In this paper we evaluate these claims, and show that all of them are unfounded. If the objective is to analyze whether mutations that modify social behavior are favored or opposed by natural selection, then no aspect of inclusive fitness theory is needed.

Engineering salt-tolerant Brassica plants: characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation.

PubMed

Zhang, H X; Hodson, J N; Williams, J P; Blumwald, E

2001-10-23

Transgenic Brassica napus plants overexpressing AtNHX1, a vacuolar Na(+)/H(+) antiport from Arabidopsis thaliana, were able to grow, flower, and produce seeds in the presence of 200 mM sodium chloride. Although the transgenic plants grown in high salinity accumulated sodium up to 6% of their dry weight, growth of the these plants was only marginally affected by the high salt concentration. Moreover, seed yields and the seed oil quality were not affected by the high salinity of the soil. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils. Our findings, showing that the modification of a single trait significantly improved the salinity tolerance of this crop plant, suggest that with a combination of breeding and transgenic plants it could be possible to produce salt-tolerant crops with far fewer target traits than had been anticipated.

Student Teachers' Attitudes and Beliefs about Inclusion and Inclusive Practice

ERIC Educational Resources Information Center

Beacham, Nigel; Rouse, Martyn

2012-01-01

The beliefs and attitudes of teachers are an important element in the development of inclusive education and its associated practices. Teacher education is seen as crucial in helping to develop positive attitudes and beliefs that are thought to promote inclusion, although attempts to reform teacher education in order to address issues of inclusion…

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

PubMed

Lin, Hsin Hung; Pan, Yih Jiuan; Hsu, Shen Hsing; Van, Ru Chuan; Hsiao, Yi Yuong; Chen, Jiun Hsien; Pan, Rong Long

2005-10-15

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

PubMed Central

Ruggiero, Melina; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.; Mojica, Maria F.; Bethel, Christopher R.; Rudin, Susan D.; Zeiser, Elise T.; Gutkind, Gabriel

2017-01-01

ABSTRACT PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 × 103 ± 0.1 × 103 M−1 s−1, where k2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k2/K range of ≈103 M−1 s−1) and not class A β-lactamases (i.e., k2/K range, 104 to 105 M−1 s−1). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k2/K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam–AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable

In-hospital outcome of transcatheter vs. surgical aortic valve replacement in patients with aortic valve stenosis: complete dataset of patients treated in 2013 in Germany.

PubMed

Möllmann, Helge; Bestehorn, Kurt; Bestehorn, Maike; Papoutsis, Konstantinos; Fleck, Eckart; Ertl, Georg; Kuck, Karl-Heinz; Hamm, Christian

2016-06-01

Transvascular (TV-AVI) or transapical (TA-AVI) aortic valve implantation (TAVI) is a treatment option for patients with aortic stenosis being at high or prohibitive risk for surgical aortic valve implantation (SAVR). Randomized data demonstrated that these subgroups can safely been treated with TAVI. However, a comparison of SAVR and TAVI in intermediate and low-risk patients is missing. Therefore, the aim of the analysis was to compare TAVI and SAVR in all patients who were treated for aortic valve stenosis in Germany throughout 1 year. The mandatory quality assurance collects data on the in-hospital outcome from all patients (n = 20,340) undergoing either SAVR or TAVI in Germany. In order to compare the different treatment approaches patients were categorized into four risk groups using the logistic EuroScore I (ES). In-hospital mortality and peri- and postprocedural complications were analyzed. The in-hospital mortality did not differ between TV-AVI and SAVR in the low risk group (ES <10 %: TV-AVI 2.4 %, SAVR 2.0 %, p = 0.302) and was significantly higher for SAVR in all other risk groups (ES 10-20 %: TV-AVI 3.5 %, SAVR 5.3 %; p = 0.025; ES 20-30 %: TV-AVI 5.5 %, SAVR 12.2 %, p < 0.001; ES >30 %: TV-AVI 6.5 %, SAVR 12.9 %, p = 0.008). TA-AVI had a significantly higher mortality in all risk groups compared to TV-AVI. In comparison to SAVR, TA-AVI had a higher mortality in patients with ES <10, comparable mortality in ES 10-20 %, and lower mortality in patients with an ES >20 %. The overall stroke rate was 2.3 %. It occurred more frequently in patients with an ES <10 % treated with a transapical approach (SAVR 1.8 %, TV-AVI 1.9 %, TA-AVI 3.1 %, p < 0.01). There were no statistically significant differences in all other comparisons. This study demonstrates that TAVI provides excellent outcomes in all risk categories. Compared with SAVR, TV-TAVI yields similar in-hospital mortality among low-risk patients and lower in

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

Index for Inclusion

ERIC Educational Resources Information Center

Smith, Allister

2005-01-01

Index for Inclusion is a programme to assist in developing learning and participation in schools. It was written by Tony Booth and Mel Ainscow from the Centre for Studies on Inclusive Education, UK. Central Normal School was pleased to have the opportunity to trial this programme.

Fluid inclusion geothermometry

USGS Publications Warehouse

Cunningham, C.G.

1977-01-01

Fluid inclusions trapped within crystals either during growth or at a later time provide many clues to the histories of rocks and ores. Estimates of fluid-inclusion homogenization temperature and density can be obtained using a petrographic microscope with thin sections, and they can be refined using heating and freezing stages. Fluid inclusion studies, used in conjunction with paragenetic studies, can provide direct data on the time and space variations of parameters such as temperature, pressure, density, and composition of fluids in geologic environments. Changes in these parameters directly affect the fugacity, composition, and pH of fluids, thus directly influencing localization of ore metals. ?? 1977 Ferdinand Enke Verlag Stuttgart.

Becoming Inclusive: A Code of Conduct for Inclusion and Diversity.

PubMed

Schmidt, Bonnie J; MacWilliams, Brent R; Neal-Boylan, Leslie

There are increasing concerns about exclusionary behaviors and lack of diversity in the nursing profession. Exclusionary behaviors, which may include incivility, bullying, and workplace violence, discriminate and isolate individuals and groups who are different, whereas inclusive behaviors encourage diversity. To address inclusion and diversity in nursing, this article offers a code of conduct. This code of conduct builds on existing nursing codes of ethics and applies to nursing students and nurses in both educational and practice settings. Inclusive behaviors that are demonstrated in nurses' relationships with patients, colleagues, the profession, and society are described. This code of conduct provides a basis for measureable change, empowerment, and unification of the profession. Recommendations, implications, and a pledge to action are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

PubMed Central

Barkla, B J; Blumwald, E

1991-01-01

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport. Images PMID:1662387

Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris.

PubMed

Barkla, B J; Blumwald, E

1991-12-15

The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions. Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity. Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly. These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange. The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide. The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations. Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity. These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

Can Ceftazidime-Avibactam and Aztreonam Overcome β-Lactam Resistance Conferred by Metallo-β-Lactamases in Enterobacteriaceae?

PubMed

Marshall, Steven; Hujer, Andrea M; Rojas, Laura J; Papp-Wallace, Krisztina M; Humphries, Romney M; Spellberg, Brad; Hujer, Kristine M; Marshall, Emma K; Rudin, Susan D; Perez, Federico; Wilson, Brigid M; Wasserman, Ronald B; Chikowski, Linda; Paterson, David L; Vila, Alejandro J; van Duin, David; Kreiswirth, Barry N; Chambers, Henry F; Fowler, Vance G; Jacobs, Michael R; Pulse, Mark E; Weiss, William J; Bonomo, Robert A

2017-04-01

Based upon knowledge of the hydrolytic profile of major β-lactamases found in Gram-negative bacteria, we tested the efficacy of the combination of ceftazidime-avibactam (CAZ-AVI) with aztreonam (ATM) against carbapenem-resistant enteric bacteria possessing metallo-β-lactamases (MBLs). Disk diffusion and agar-based antimicrobial susceptibility testing were initially performed to determine the in vitro efficacy of a unique combination of CAZ-AVI and ATM against 21 representative Enterobacteriaceae isolates with a complex molecular background that included bla IMP , bla NDM , bla OXA-48 , bla CTX-M , bla AmpC , and combinations thereof. Time-kill assays were conducted, and the in vivo efficacy of this combination was assessed in a murine neutropenic thigh infection model. By disk diffusion assay, all 21 isolates were resistant to CAZ-AVI alone, and 19/21 were resistant to ATM. The in vitro activity of CAZ-AVI in combination with ATM against diverse Enterobacteriaceae possessing MBLs was demonstrated in 17/21 isolates, where the zone of inhibition was ≥21 mm. All isolates demonstrated a reduction in CAZ-AVI agar dilution MICs with the addition of ATM. At 2 h, time-kill assays demonstrated a ≥4-log 10 -CFU decrease for all groups that had CAZ-AVI with ATM (8 μg/ml) added, compared to the group treated with CAZ-AVI alone. In the murine neutropenic thigh infection model, an almost 4-log 10 -CFU reduction was noted at 24 h for CAZ-AVI (32 mg/kg every 8 h [q8h]) plus ATM (32 mg/kg q8h) versus CAZ-AVI (32 mg/kg q8h) alone. The data presented herein require us to carefully consider this new therapeutic combination to treat infections caused by MBL-producing Enterobacteriaceae . Copyright © 2017 American Society for Microbiology.

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination1

PubMed Central

Cooley, Michael B.; Yang, Hong; Dahal, Peetambar; Mella, R. Alejandra; Downie, A. Bruce; Haigh, Anthony M.; Bradford, Kent J.

1999-01-01

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA4+7. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V0 membrane sector of vacuolar H+-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V1 sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds. PMID:10594121

Fluid inclusions in stony meteorites

NASA Technical Reports Server (NTRS)

Warner, J. L.; Ashwal, L. D.; Bergman, S. C.; Gibson, E. K., Jr.; Henry, D. J.; Lee-Berman, R.; Roedder, E.; Belkin, H. E.

1983-01-01

The fluid inclusions presently described for five stony meteorites brings to seven the number of such meteorites confirmed. Homogenization temperatures are reproducible in each inclusion, and range from 25 C to over 225 C, with some vapor plus liquid inclusions remaining at 225 C, the highest temperature in these microthermometric experiments. Upon cooling, the fluid in some inclusions appears to freeze, as indicated by deformation and immobilization of the vapor bubble at low temperatures. Melting temperatures are by contrast difficult to observe and are not reproducible. Microthermometric data for the fluid in diogenite ALPHA 77256 and inclusions in four chondrites suggest that the fluid is aqueous, with a high solute content.

Loss of G2 subunit of vacuolar-type proton transporting ATPase leads to G1 subunit upregulation in the brain

PubMed Central

Kawamura, Nobuyuki; Sun-Wada, Ge-Hong; Wada, Yoh

2015-01-01

Vacuolar-type ATPase (V-ATPase) is a primary proton pump with versatile functions in various tissues. In nerve cells, V-ATPase is required for accumulation of neurotransmitters into secretory vesicles and subsequent release at the synapse. Neurons express a specific isoform (G2) of the G subunit of V-ATPase constituting the catalytic sector of the enzyme complex. Using gene targeting, we generated a mouse lacking functional G2 (G2 null), which showed no apparent disorders in architecture and behavior. In the G2-null mouse brain, a G1 subunit isoform, which is ubiquitously expressed in neuronal and non-neuronal tissues, accumulated more abundantly than in wild-type animals. This G1 upregulation was not accompanied by an increase in mRNA. These results indicate that loss of function of neuron-specific G2 isoform was compensated by an increase in levels of the G1 isoform without apparent upregulation of the G1 mRNA. PMID:26353914

Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed.

PubMed

Kumamaru, Toshihiro; Uemura, Yuji; Inoue, Yoshimi; Takemoto, Yoko; Siddiqui, Sadar Uddin; Ogawa, Masahiro; Hara-Nishimura, Ikuko; Satoh, Hikaru

2010-01-01

To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus.

PubMed

Giner, Ana; Pascual, Laura; Bourgeois, Michael; Gyetvai, Gabor; Rios, Pablo; Picó, Belén; Troadec, Christelle; Bendahmane, Abdel; Garcia-Mas, Jordi; Martín-Hernández, Ana Montserrat

2017-09-05

In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem.

Palmitate-Induced Vacuolar-Type H+-ATPase Inhibition Feeds Forward Into Insulin Resistance and Contractile Dysfunction.

PubMed

Liu, Yilin; Steinbusch, Laura K M; Nabben, Miranda; Kapsokalyvas, Dimitris; van Zandvoort, Marc; Schönleitner, Patrick; Antoons, Gudrun; Simons, Peter J; Coumans, Will A; Geomini, Amber; Chanda, Dipanjan; Glatz, Jan F C; Neumann, Dietbert; Luiken, Joost J F P

2017-06-01

Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H + -ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V 1 and the integral membrane V 0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction. © 2017 by the American Diabetes Association.

Nanotubular Toughening Inclusions

NASA Technical Reports Server (NTRS)

Park, Cheol (Inventor); Working, Dennis C. (Inventor); Siochi, Emilie J. (Inventor); Harrison, Joycelyn S. (Inventor)

2017-01-01

Conventional toughening agents are typically rubbery materials or small molecular weight molecules, which mostly sacrifice the intrinsic properties of a matrix such as modulus, strength, and thermal stability as side effects. On the other hand, high modulus inclusions tend to reinforce elastic modulus very efficiently, but not the strength very well. For example, mechanical reinforcement with inorganic inclusions often degrades the composite toughness, encountering a frequent catastrophic brittle failure triggered by minute chips and cracks. Thus, toughening generally conflicts with mechanical reinforcement. Carbon nanotubes have been used as efficient reinforcing agents in various applications due to their combination of extraordinary mechanical, electrical, and thermal properties. Moreover, nanotubes can elongate more than 20% without yielding or breaking, and absorb significant amounts of energy during deformation, which enables them to also be an efficient toughening agent, as well as excellent reinforcing inclusion. Accordingly, an improved toughening method is provided by incorporating nanotubular inclusions into a host matrix, such as thermoset and thermoplastic polymers or ceramics without detrimental effects on the intrinsic physical properties of the matrix.

Nanotubular Toughening Inclusions

NASA Technical Reports Server (NTRS)

Park, Cheol (Inventor); Working, Dennis C. (Inventor); Siochi, Emilie J. (Inventor); Harrison, Joycelyn S. (Inventor)

2015-01-01

Conventional toughening agents are typically rubbery materials or small molecular weight molecules, which mostly sacrifice the intrinsic properties of a matrix such as modulus, strength, and thermal stability as side effects. On the other hand, high modulus inclusions tend to reinforce elastic modulus very efficiently, but not the strength very well. For example, mechanical reinforcement with inorganic inclusions often degrades the composite toughness, encountering a frequent catastrophic brittle failure triggered by minute chips and cracks. Thus, toughening generally conflicts with mechanical reinforcement. Carbon nanotubes have been used as efficient reinforcing agents in various applications due to their combination of extraordinary mechanical, electrical, and thermal properties. Moreover, nanotubes can elongate more than 20% without yielding or breaking, and absorb significant amounts of energy during deformation, which enables them to also be an efficient toughening agent, as well as excellent reinforcing inclusion. Accordingly, an improved toughening method is provided by incorporating nanotubular inclusions into a host matrix, such as thermoset and thermoplastic polymers or ceramics without detrimental effects on the matrix's intrinsic physical properties.

The effect of auditory verbal imagery on signal detection in hallucination-prone individuals

PubMed Central

Moseley, Peter; Smailes, David; Ellison, Amanda; Fernyhough, Charles

2016-01-01

Cognitive models have suggested that auditory hallucinations occur when internal mental events, such as inner speech or auditory verbal imagery (AVI), are misattributed to an external source. This has been supported by numerous studies indicating that individuals who experience hallucinations tend to perform in a biased manner on tasks that require them to distinguish self-generated from non-self-generated perceptions. However, these tasks have typically been of limited relevance to inner speech models of hallucinations, because they have not manipulated the AVI that participants used during the task. Here, a new paradigm was employed to investigate the interaction between imagery and perception, in which a healthy, non-clinical sample of participants were instructed to use AVI whilst completing an auditory signal detection task. It was hypothesized that AVI-usage would cause participants to perform in a biased manner, therefore falsely detecting more voices in bursts of noise. In Experiment 1, when cued to generate AVI, highly hallucination-prone participants showed a lower response bias than when performing a standard signal detection task, being more willing to report the presence of a voice in the noise. Participants not prone to hallucinations performed no differently between the two conditions. In Experiment 2, participants were not specifically instructed to use AVI, but retrospectively reported how often they engaged in AVI during the task. Highly hallucination-prone participants who retrospectively reported using imagery showed a lower response bias than did participants with lower proneness who also reported using AVI. Results are discussed in relation to prominent inner speech models of hallucinations. PMID:26435050

Removal of inclusions from silicon

NASA Astrophysics Data System (ADS)

Ciftja, Arjan; Engh, Thorvald Abel; Tangstad, Merete; Kvithyld, Anne; Øvrelid, Eivind Johannes

2009-11-01

The removal of inclusions from molten silicon is necessary to satisfy the purity requirements for solar grade silicon. This paper summarizes two methods that are investigated: (i) settling of the inclusions followed by subsequent directional solidification and (infiltration by ceramic foam filters. Settling of inclusions followed by directional solidification is of industrial importance for production of low-cost solar grade silicon. Filtration is reported as the most efficient method for removal of inclusions from the top-cut silicon scrap.

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

Lending a Helping Hand at Work: A Multilevel Investigation of Prosocial Motivation, Inclusive Climate and Inclusive Behavior.

PubMed

Nelissen, Philippe T J H; Hülsheger, Ute R; van Ruitenbeek, Gemma M C; Zijlstra, Fred R H

2017-09-01

Purpose People with disabilities often encounter difficulties at the workplace such as exclusion or unfair treatment. Researchers have therefore pointed to the need to focus on behavior that fosters inclusion as well as variables that are antecedents of such 'inclusive behavior'. Therefore the purpose of this study was to research the relationship between prosocial motivation, team inclusive climate and employee inclusive behavior. Method A survey was conducted among a sample of 282 paired employees and colleagues, which were nested in 84 teams. Employees self-rated prosocial motivation and team inclusive climate, their inclusive behavior was assessed by colleagues. Hypotheses were tested using multilevel random coefficient modeling. Results Employees who are prosocially motivated will display more inclusive behavior towards people with disabilities, and this relationship is moderated by team inclusive climate in such a way that the relationship is stronger when the inclusive climate is high. Conclusion This study shows that inclusive organizations, which value a diverse workforce, need to be aware of not only individual employee characteristics, but also team level climate to ensure the smooth integrations of people with disabilities into regular work teams.

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase.

PubMed

Ruggiero, Melina; Papp-Wallace, Krisztina M; Taracila, Magdalena A; Mojica, Maria F; Bethel, Christopher R; Rudin, Susan D; Zeiser, Elise T; Gutkind, Gabriel; Bonomo, Robert A; Power, Pablo

2017-06-01

PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 / K = 2 × 10 3 ± 0.1 × 10 3 M -1 s -1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2 / K range of ≈10 3 M -1 s -1 ) and not class A β-lactamases (i.e., k 2 / K range, 10 4 to 10 5 M -1 s -1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation ( k 2 / K ) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable

Diverse Perspectives on Inclusive School Communities

ERIC Educational Resources Information Center

Tsokova, Diana; Tarr, Jane

2012-01-01

What is an inclusive school community? How do stakeholders perceive their roles and responsibilities towards inclusive school communities? How can school communities become more inclusive through engagement with individual perspectives? "Diverse Perspectives on Inclusive School Communities" captures and presents the voices of a wide…

A review of social inclusion measures.

PubMed

Coombs, Tim; Nicholas, Angela; Pirkis, Jane

2013-10-01

Social inclusion is crucial to mental health and well-being and is emphasised in Australia's Fourth National Mental Health Plan. There is a recognition that a measure of social inclusion would complement the suite of outcome measures that is currently used in public sector mental health services. This paper is an initial scope of candidate measures of social inclusion and considers their suitability for this purpose. We identified potential measures through searches of PsycINFO and Medline and a more general Internet search. We extracted descriptive and evaluative information on each measure identified and compared this information with a set of eight criteria. The criteria related to the measure's inclusion of four domains of social inclusion outlined in Australia's Fourth National Mental Health Plan, its usability within the public mental health sector and its psychometric properties. We identified 10 candidate measures of social inclusion: the Activity and Participation Questionnaire (APQ-6); the Australian Community Participation Questionnaire (ACPQ); the Composite Measure of Social Inclusion (CMSI); the EMILIA Project Questionnaire (EPQ); the Evaluating Social Inclusion Questionnaire (ESIQ); the Inclusion Web (IW); the Social and Community Opportunities Profile (SCOPE); the Social Inclusion Measure (SIM); the Social Inclusion Questionnaire (SIQ); and the Staff Survey of Social Inclusion (SSSI). After comparison with the eight review criteria, we determined that the APQ-6 and the SCOPE-short form show the most potential for further testing. Social inclusion is too important not to measure. This discussion of individual-level measures of social inclusion provides a springboard for selecting an appropriate measure for use in public sector mental health services. It suggests that there are two primary candidates, but neither of these is quite fit-for-purpose in their current form. Further exploration will reveal whether one of these is suitable, whether another

Exploring Preservice Teachers' Attitudes Towards Inclusion

ERIC Educational Resources Information Center

Killoran, Isabel; Woronko, Dagmara; Zaretsky, Hayley

2014-01-01

This study responds to a call for research into existing teacher-education programmes and their impact on teacher candidates' attitudes. An inclusive education course that examined the difference between "soft inclusion" (inclusion which addresses the issue of place rather than substance of learning) and genuine inclusion was used to…

Shock Re-equilibration of Fluid Inclusions

NASA Technical Reports Server (NTRS)

Madden, M. E. Elwood; Horz, F.; Bodnar, R. J.

2004-01-01

Fluid inclusions (microscopic volumes of fluid trapped within minerals as they precipitate) are extremely common in terrestrial minerals formed under a wide range of geological conditions from surface evaporite deposits to kimberlite pipes. While fluid inclusions in terrestrial rocks are nearly ubiquitous, only a few fluid inclusion-bearing meteorites have been documented. The scarcity of fluid inclusions in meteoritic materials may be a result of (a) the absence of fluids when the mineral was formed on the meteorite parent body or (b) the destruction of fluid inclusions originally contained in meteoritic materials by subsequent shock metamorphism. However, the effects of impact events on pre-existing fluid inclusions trapped in target and projectile rocks has received little study. Fluid inclusions trapped prior to the shock event may be altered (re-equilibrated) or destroyed due to the high pressures, temperatures, and strain rates associated with impact events. By examining the effects of shock deformation on fluid inclusion properties and textures we may be able to better constrain the pressure-temperature path experienced by terrestrial and meteoritic shocked materials and also gain a clearer understanding of why fluid inclusions are rarely found in meteorite samples.

Inclusive Education under Collectivistic Culture

ERIC Educational Resources Information Center

Futaba, Yasuko

2016-01-01

This paper addresses how inclusive education under collective culture is possible. Inclusive education, which more-or-less involves changing the current schools, has been denied, doubted or distorted by both policy-makers and practitioners of general and special education in Japan. Main reason for the setback in inclusive education can be…

Fluid Inclusion Gas Analysis

DOE Data Explorer

Dilley, Lorie

2013-01-01

Fluid inclusion gas analysis for wells in various geothermal areas. Analyses used in developing fluid inclusion stratigraphy for wells and defining fluids across the geothermal fields. Each sample has mass spectrum counts for 180 chemical species.

Inclusive Education in South Korea

ERIC Educational Resources Information Center

Kim, Yong-Wook

2014-01-01

The purpose of this paper is to examine the current implementation of inclusive education in South Korea and discuss its challenges. The history of special education is first described followed by an introduction to policies relevant to special and inclusive education. Next, a critical discussion of the state of inclusive education follows built…

Inclusion in High-Achieving Singapore: Challenges of Building an Inclusive Society in Policy and Practice

ERIC Educational Resources Information Center

Walker, Zachary; Musti-Rao, Shobana

2016-01-01

Building an inclusive society in which all people can participate effectively and live together requires understanding inclusive education and its impact on the social order. As countries of different regions face the vast array of challenges unique to their educational systems, it becomes apparent that inclusive societies are intricately tied to…

Crack-Inclusion Interaction: A Review

DTIC Science & Technology

2014-03-01

research on the problem. fracture mechanics, inclusion, crack, dislocation, Erdogan , Dundurs, integral equation, Green’s function 58 Christopher S. Meyer...14 Figure 9. Geometry for the crack-inclusion problem, adapted from Erdogan and Wei (21) . . . 23 Figure 10. The...crack-inclusion problem geometry, adapted from Erdogan and Wei (21): figure 9 is repeated here in the text for convenience

Socially Inclusive Pedagogy in Literacy Classes: Fostering Inclusion in the Inner City

ERIC Educational Resources Information Center

Cleovoulou, Yiola

2008-01-01

Drawing on case studies of five elementary school teachers in one inner city school, the author explored ways teachers foster social inclusion in their classrooms. Rooted in classroom observations and extensive teacher interviews, teachers' teaching methods and practices were examined as a base from which to explore socially inclusive pedagogy in…

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

PubMed Central

de Vries, Michel

2016-01-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. PMID:26977085

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

PubMed

Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

Resources on Inclusive Education.

ERIC Educational Resources Information Center

Hulgin, Kathy, Ed.

This information packet is intended for use by teachers, parents, administrators, therapists, advocates, and others interested in achieving inclusive educational opportunities for students with disabilities. Part I provides reprints of selected readings, including: "Sample Case Studies of Inclusive Education" (Janet Duncan); "Public Schools…

Using melt inclusions and fluid inclusions to track ore-metal behavior in magma-hydrothermal systems (Invited)

NASA Astrophysics Data System (ADS)

Lowenstern, J. B.; Audétat, A.

2013-12-01

Melt and fluid inclusions yield important clues to the history of igneous melts and their related hydrothermal ore deposits (1). Under ideal conditions, melt inclusions in volcanic rocks yield data on the actual concentrations of ore metals and volatiles during instantaneous snapshots of crystallization and degassing. Their varying compositions can directly reflect sequestration of ore-metals in fractionating minerals and/or exsolving brines and vapors. Frequently, scientists compare the concentration of volatile elements in melt inclusions with their abundance in devolatilized matrix glass. Though this provides an informative qualitative overview of volatility, it is essentially impossible to use such data to calculate thermodynamically relevant partition coefficients. The resulting partitioning ratio instead represents fractionation over a wide range of pressures, and compositions (for both exsolved fluid and silicate melt). Ideally, workers should identify co-entrapped fluid and glass inclusions to provide more thermodynamically meaningful partitioning ratios for volatile metals and gases (2,3). Unfortunately, the occurrence of fluid inclusions co-entrapped with silicate melt is relatively rare, and studies of synthetic fluid and melt inclusions may be the most practical means of exploring the effect of crystallization and degassing in 'natural' systems. As with melt inclusions, under ideal conditions, fluid inclusions in intrusive rocks represent the compositions of fluids generated within associated magmatic-hydrothermal fluid systems. Multiple generations of cross-cutting fractures may be generated, resulting in trails of secondary and pseudosecondary inclusions in igneous minerals, and primary and secondary inclusions in hydrothermal assemblages. Chemistry of the fluids preserved within different inclusion generations will change markedly due to changes in magmatic temperature and pressure and mixing of diverse external fluids from meteoric and metamorphic

Melt inclusions: Chapter 6

USGS Publications Warehouse

,; Lowenstern, J. B.

2014-01-01

Melt inclusions are small droplets of silicate melt that are trapped in minerals during their growth in a magma. Once formed, they commonly retain much of their initial composition (with some exceptions) unless they are re-opened at some later stage. Melt inclusions thus offer several key advantages over whole rock samples: (i) they record pristine concentrations of volatiles and metals that are usually lost during magma solidification and degassing, (ii) they are snapshots in time whereas whole rocks are the time-integrated end products, thus allowing a more detailed, time-resolved view into magmatic processes (iii) they are largely unaffected by subsolidus alteration. Due to these characteristics, melt inclusions are an ideal tool to study the evolution of mineralized magma systems. This chapter first discusses general aspects of melt inclusions formation and methods for their investigation, before reviewing studies performed on mineralized magma systems.

Mechanism of development of ionocytes rich in vacuolar-type H+-ATPase in the skin of zebrafish larvae

PubMed Central

Esaki, Masahiro; Hoshijima, Kazuyuki; Nakamura, Nobuhiro; Munakata, Keijiro; Tanaka, Mikiko; Ookata, Kayoko; Asakawa, Kazuhide; Kawakami, Koichi; Wang, Weiyi; Weinberg, Eric S.; Hirose, Shigehisa

2009-01-01

Mitochondrion-rich cells (MRCs), or ionocytes, play a central role in aquatic species, maintaining body fluid ionic homeostasis by actively taking up or excreting ions. Since their first description in 1932 in eel gills, extensive morphological and physiological analyses have yielded important insights into ionocyte structure and function, but understanding the developmental pathway specifying these cells remains an ongoing challenge. We previously succeeded in identifying a key transcription factor, Foxi3a, in zebrafish larvae by database mining. In the present study, we analyzed a zebrafish mutant, quadro (quo), deficient in foxi1 gene expression and found that foxi1 is essential for development of an MRC subpopulation rich in vacuolar-type H+-ATPase (vH-MRC). foxi1 acts upstream of Delta-Notch signaling that determines sporadic distribution of vH-MRC and regulates foxi3a expression. Through gain- and loss-of-function assays and cell transplantation experiments, we further clarified that (1) the expression level of foxi3a is maintained by a positive feedback loop between foxi3a and its downstream gene gcm2 and (2) Foxi3a functions cell-autonomously in the specification of vH-MRC. These observations provide a better understanding of the differentiation and distribution of the vH-MRC subtype. PMID:19268451

The German Aortic Valve Registry (GARY): in-hospital outcome

PubMed Central

Hamm, Christian W.; Möllmann, Helge; Holzhey, David; Beckmann, Andreas; Veit, Christof; Figulla, Hans-Reiner; Cremer, J.; Kuck, Karl-Heinz; Lange, Rüdiger; Zahn, Ralf; Sack, Stefan; Schuler, Gerhard; Walther, Thomas; Beyersdorf, Friedhelm; Böhm, Michael; Heusch, Gerd; Funkat, Anne-Kathrin; Meinertz, Thomas; Neumann, Till; Papoutsis, Konstantinos; Schneider, Steffen; Welz, Armin; Mohr, Friedrich W.

2014-01-01

Background Aortic stenosis is a frequent valvular disease especially in elderly patients. Catheter-based valve implantation has emerged as a valuable treatment approach for these patients being either at very high risk for conventional surgery or even deemed inoperable. The German Aortic Valve Registry (GARY) provides data on conventional and catheter-based aortic procedures on an all-comers basis. Methods and results A total of 13 860 consecutive patients undergoing repair for aortic valve disease [conventional surgery and transvascular (TV) or transapical (TA) catheter-based techniques] have been enrolled in this registry during 2011 and baseline, procedural, and outcome data have been acquired. The registry summarizes the results of 6523 conventional aortic valve replacements without (AVR) and 3464 with concomitant coronary bypass surgery (AVR + CABG) as well as 2695 TV AVI and 1181 TA interventions (TA AVI). Patients undergoing catheter-based techniques were significantly older and had higher risk profiles. The stroke rate was low in all groups with 1.3% (AVR), 1.9% (AVR + CABG), 1.7% (TV AVI), and 2.3% (TA AVI). The in-hospital mortality was 2.1% (AVR) and 4.5% (AVR + CABG) for patients undergoing conventional surgery, and 5.1% (TV AVI) and AVI 7.7% (TA AVI). Conclusion The in-hospital outcome results of this registry show that conventional surgery yields excellent results in all risk groups and that catheter-based aortic valve replacements is an alternative to conventional surgery in high risk and elderly patients. PMID:24022003

Innovation for an Inclusive Future

NASA Astrophysics Data System (ADS)

Springett, Mark; Rice, Mark; Carmichael, Alex; Griffiths, Richard

This workshop will focus on setting the agenda for research, practice and policy in support of inclusive design for third generation computer-based products. The next generation of technology represents an unprecedented opportunity to improve the quality of life for groups of users who have previously faced exclusion, such as those with impairments and older citizens. At the same time it risks creating a greater digital divide and further exclusion. How we approach design for this new generation will determine whether or not the third wave will provide positive advances towards an inclusive digital world. We therefore need to put forward both a rationale for inclusive design and provide pointers towards technical development and design practice in support of inclusion. It is our belief that there is not only a strong moral case for design for inclusion but also significant commercial incentive, which may be key to persuading influential players to focus on inclusion. Therefore one of our key objectives is to describe and promote the advantages of designing ‘in from the edges’ of the user population rather than designing for a notional ‘average’ user.

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

PubMed

Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

2016-03-31

Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

Inclusive Services Innovation Configuration

ERIC Educational Resources Information Center

Holdheide, Lynn R.; Reschly, Daniel J.

2011-01-01

Teacher preparation to deliver inclusive services to students with disabilities is increasingly important because of changes in law and policy emphasizing student access to, and achievement in, the general education curriculum. This innovation configuration identifies the components of inclusive services that should be incorporated in teacher…

Transcatheter aortic valve implantation transapical: step by step.

PubMed

Walther, Thomas; Möllmann, Helge; van Linden, Arnaud; Kempfert, Jörg

2011-01-01

Transcatheter aortic valve implantation (T-AVI) has been introduced into clinical practice to treat high-risk elderly patients with aortic stenosis. T-AVI can be performed by using a retrograde transfemoral (TF), transsubclavian, transaortic, and/or antegrade transapical (TA) approach. For TA-AVI, CE mark approval was granted in 2008 for the Edwards SAPIEN (Edwards Lifesciences, Irvine, CA) prosthesis with the Ascendra delivery system and in 2010 for the second-generation Edwards SAPIEN XT prosthesis and the Ascendra II delivery system, with 23-mm and 26-mm valves. In 2011, CE mark approval has been granted for TA-AVI by using the SAPIEN XT 29-mm prosthesis. Several other devices from different companies (Jenavalve, Jena Valve Inc, Munich, Germany; Embracer, Medtronic Inc, Guilford, CT; Accurate, Symetis Inc, Geneva, Switzerland) have passed "first in man trials" successfully and are being evaluated within multicenter pivotal studies. In this article we will focus on specific aspects of the TA technique for AVI. Copyright © 2011 Elsevier Inc. All rights reserved.

When Inclusion Is Innovation: An Examination of Administrator Perspectives on Inclusion in China

ERIC Educational Resources Information Center

Hu, Bi Ying; Roberts, Sherron Killingsworth

2011-01-01

This article examines administrator perspectives of innovative services for the inclusion of young children with disabilities in regular preschool classrooms in China. Twelve directors from 12 pilot inclusion preschools in Beijing participated in this study. Qualitative interview results revealed the following subthemes: definition, advocacy,…

Gene-Specific Countermeasures Against Ebola Virus Based on Antisense Phosphorodiamidate Morpholino Oligomers

DTIC Science & Technology

2006-01-01

Bavari 1* 1 US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland, United States of America, 2 AVI BioPharma ...used as controls in these experiments. The PMOs were synthesized by AVI BioPharma , (Corvallis, Oregon, United States), as previously described [35]. In...interests. RB, DAS, and PLI have financial interest in AVI BioPharma , the company that supplied the PMO compounds for these studies. & References 1

VP35 Knockdown Inhibits Ebola Virus Amplification and Protects Against Lethal Infection in Mice

DTIC Science & Technology

2006-03-01

Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702-50112; and AVI BioPharma Inc., Corvallis, Oregon3 Received 29 June...target nucleotides within the EBOV genome or antigenome (Table 1). All PMO were synthesized and purified by AVI BioPharma Inc. (Cor- vallis, OR), as...technical assistance. We are also grateful to the Chemistry Department at AVI BioPharma Inc. for expert synthesis, purification, and analysis of all PMO

Embracing Inclusive Teacher Education

ERIC Educational Resources Information Center

Moran, Anne

2007-01-01

This paper examines the extent to which initial teacher education (ITE) programmes contribute to the development of inclusive attitudes, values and practices. Inclusive education is the entitlement of all children and young people to quality education, irrespective of their differences or dispositions. It is about embracing educational values of…

Geometric Modeling of Inclusions as Ellipsoids

NASA Technical Reports Server (NTRS)

Bonacuse, Peter J.

2008-01-01

Nonmetallic inclusions in gas turbine disk alloys can have a significant detrimental impact on fatigue life. Because large inclusions that lead to anomalously low lives occur infrequently, probabilistic approaches can be utilized to avoid the excessively conservative assumption of lifing to a large inclusion in a high stress location. A prerequisite to modeling the impact of inclusions on the fatigue life distribution is a characterization of the inclusion occurrence rate and size distribution. To help facilitate this process, a geometric simulation of the inclusions was devised. To make the simulation problem tractable, the irregularly sized and shaped inclusions were modeled as arbitrarily oriented, three independent dimensioned, ellipsoids. Random orientation of the ellipsoid is accomplished through a series of three orthogonal rotations of axes. In this report, a set of mathematical models for the following parameters are described: the intercepted area of a randomly sectioned ellipsoid, the dimensions and orientation of the intercepted ellipse, the area of a randomly oriented sectioned ellipse, the depth and width of a randomly oriented sectioned ellipse, and the projected area of a randomly oriented ellipsoid. These parameters are necessary to determine an inclusion s potential to develop a propagating fatigue crack. Without these mathematical models, computationally expensive search algorithms would be required to compute these parameters.

Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2 *

PubMed Central

Diab, Heba I.; Kane, Patricia M.

2013-01-01

Vacuolar H+-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125–7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (PFIT2-GFP) or the TSA2 promoter (PTSA2-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated PFIT2-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased PFIT2-GFP expression and, surprisingly, restored PTSA2-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and PFIT2-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis. PMID:23457300

Loss of vacuolar H+-ATPase (V-ATPase) activity in yeast generates an iron deprivation signal that is moderated by induction of the peroxiredoxin TSA2.

PubMed

Diab, Heba I; Kane, Patricia M

2013-04-19

Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.

Inclusive Schools. Topical Bibliography on Inclusive Schools.

ERIC Educational Resources Information Center

Sorenson, Barbara, Comp.; Drill, Janet, Comp.

This abstract bibliography of approximately 200 references looks at various aspects of inclusive schools. References are a result of computer searches of three databases: the Educational Resources Information Center (ERIC), Exceptional Child Education Resources, and the Western Regional Resources Center. Preliminary information includes directions…

Sliding inclusions and their applications

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

Mura, T.

It is found that when an ellipsoidal inclusion undergoes a shear eigenstrain and the inclusion is free to slip along the interface, the stress field vanishes everywhere in the inclusion and the matrix. It is assumed in the analysis that the inclusion interface cannot sustain any shear traction. There exists a shear deformation which transforms an ellipsoid into the identical ellipsoid without changing its orientation (ellipsoid invariant transformation). Therefore, no resistance for shear deformation is expected. This may be a characteristic of deformation seen in superplasticity alloys and granular materials. The theory is valid even for large deformations when incrementalmore » strains (or strain rates) are considered instead of strains themselves.« less

Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development.

PubMed

Leskow, Carla Coluccio; Kamenetzky, Laura; Dominguez, Pia Guadalupe; Díaz Zirpolo, José Antonio; Obata, Toshihiro; Costa, Hernán; Martí, Marcelo; Taboga, Oscar; Keurentjes, Joost; Sulpice, Ronan; Ishihara, Hirofumi; Stitt, Mark; Fernie, Alisdair Robert; Carrari, Fernando

2016-07-01

Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Detecting and Correcting Melt Inclusion Modification

NASA Astrophysics Data System (ADS)

Cottrell, E.; Kelley, K. A.

2008-12-01

Post entrapment diffusive modification of melt inclusions may mute or erase primary signatures. Corrections for post-entrapment crystallization (PEC) and Fe-loss are routinely applied and, because recent experimental studies suggest rapid diffusion of trace components into and out of olivine-hosted inclusions, the ability to discriminate between primary and secondary signatures is now even more critical. Two tools may assist in this endeavor. XANES measurements of Fe3+/ΣFe ratios in undegassed ol-hosted basaltic melt inclusions from global arcs are 16-36% (n=16), significantly higher than the 7-10% commonly assumed, and higher than in MORB or BABB lavas (Kelley and Cottrell, this mtg). The Fe3+/ΣFe ratios indicate melt-host equilibrium, with significantly less PEC or Fe-loss than would have been otherwise assumed. We conclude that Fe2+ diffusion has been minimal; therefore the residence time of these primitive inclusions in an evolved magma must have been short. Fe3+/ΣFe correlates positively with water concentration, but not with CO2 and S concentrations or Mg#. The oxidized nature of arc lavas and melt inclusions may therefore indicate an oxidized source rather than late-stage degassing or fractionation. Trace element concentrations evolve with time if an inclusion is out of equilibrium with its host. The numerical model of Cottrell et al., 2002, makes specific predictions about how suites of melt inclusions evolve, creating a tool to detect post-entrapment modification. Recent laboratory measurements of REE diffusion in olivine greatly diverge (at 1300°C, 1015 vs 1019m2/s). If REE diffusivity is extremely fast, melt inclusion HREE diversity shouldn't survive more than a few years in a magma chamber; but if slow, HREE variance could be preserved for >104 yrs. Model analysis of published suites of ol-hosted inclusions indicates that either REE diffusion is quite slow, or the residence time of melt inclusions at high temperature is very short. Loss of variance

Supporting Students with Severe Disabilities in Inclusive Schools: A Descriptive Account From Schools Implementing Inclusive Practices

ERIC Educational Resources Information Center

Kurth, Jennifer A.; Lyon, Kristin J.; Shogren, Karrie A.

2015-01-01

The purpose of the present study was to investigate practices that support the inclusion of students with severe disabilities in the learning and social activities of inclusive K-8 schools to inform inclusive school reform research and practice. Eighteen K-8 students with severe disabilities in six schools recognized for their implementation of…

Inclusive Education: Identifying Teachers' Strategies for Coping with Perceived Stressors in Inclusive Classrooms

ERIC Educational Resources Information Center

Brackenreed, Darlene

2011-01-01

This research replicates the study conducted by Forlin (2001) in Churchlands, Western Australia. Forlin's Inclusive Education "Teacher Stress and Coping Questionnaire" was adapted from the original questionnaire to more accurately reflect the language and practice of inclusion in Ontario. The purpose of this portion of the study was to…

School Inclusion Programmes (SIPS)

ERIC Educational Resources Information Center

Drossinou-Korea, Maria; Matousi, Dimitra; Panopoulos, Nikolaos; Paraskevopoulou, Aikaterini

2016-01-01

The purpose of this work was to understand the school inclusion programmes (SIPs) for students with special educational needs (SEN). The methodology was conducted in the field of special education (SE) and focuses on three case studies of students who was supported by SIPs. The Targeted, Individual, Structured, Inclusion Programme for students…

Gene expression of H+-pumps in plasma and vacuolar membranes of corn root cells under the effect of sodium ions and bioactive preparations.

PubMed

Kovalenko, N O; Palladina, T A

2016-01-01

Four isoforms of H+-ATPase of plasma membrane: MHA1, MHA2, MHA3, MHA4 are expressed in the corn seedling roots with prevalence of genes MHA3 і MHA4. The exposure of seedlings in the presence of 0.1 M NaCl activated the expression of MHA4 gene isoform, that demonstrates its important role in the processes of adaptation to salinization conditions. In vacuolar membrane, where potential is created by two Н+-pumps, sodium ions activated gene expression of only Н+-АТРase of V-type, taking no effect on the expression of Н+-pyrophosphatase. The seeds pretreatment by synthetic preparations Methyure and Ivine did not affect gene expression of Н+-pumps. Thus we can suppose that the ability of the above preparations to activate functioning of Н+-pumps in the presence of sodium ions is realized at the post-tranlation level.

Frictional sliding inclusions

NASA Astrophysics Data System (ADS)

Huang, Jin H.; Furuhashi, R.; Mura, T.

1993-02-01

S OLUTIONS ARE presented in closed form by using an averaging method for inclusions sliding along an interface due to uniform eigenstrains precribed in the inclusions. The associated stress fields are also analytically determined. A parameter s is introduced to indicate the relative magnitude of sliding compared with the extreme cases of perfect bonding and perfect sliding. When the parameter s becomes zero, the present solution coincides with Eshelby's solution which is the perfectly bonded case. In contrast, when the parameter s is unity, the solution agrees with Volterra's solution (M URA and F URUHASHI, 1984, J. appl. Mech.51, 308] for the perfect sliding case. Because of non-uniform elastic fields caused by sliding along the interface, the well-known Eshelby tensor is modified for the sliding inclusions. Moreover, based on the Mori-Tanaka theory (M ORI and T ANAKA, 1973, Acta Metall.21, 571), an overall stress-strain relation is established to characterize the sliding effect on the overall elastic moduli.

Inclusion Body Myositis

PubMed Central

Dimachkie, Mazen M.; Barohn, Richard J.

2012-01-01

The idiopathic inflammatory myopathies are a group of rare disorders that share many similarities. These include dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM), and sporadic inclusion body myositis (IBM). Inclusion body myositis is the most common idiopathic inflammatory myopathy after age 50 and it presents with chronic proximal leg and distal arm asymmetric mucle weakness. Despite similarities with PM, it is likely that IBM is primarily a degenerative disorder rather than an inflammatory muscle disease. Inclusion body myositis is associated with a modest degree of creatine kinase (CK) elevation and an abnormal electromyogram demonstrating an irritative myopathy with some chronicity. The muscle histopathology demonstrates inflammatory exudates surrounding and invading nonnecrotic muscle fibers often times accompanied by rimmed vacuoles. In this chapter, we review sporadic IBM. We also examine past, essentially negative, clinical trials in IBM and review ongoing clinical trials. For further details on DM, PM, and NM, the reader is referred to the idiopathic inflammatory myopathies chapter. PMID:23117948

Inclusive outreach practices in Palaeontology: Inclusive-Coworking

NASA Astrophysics Data System (ADS)

García-Frank, Alejandra; Gomez-Heras, Miguel; Fesharaki, Omid

2017-04-01

Previous experiences with people with both physical and intellectual functional diversity around palaeontological issues have demonstrated the important value of science outreach directed to people with disabilities. The aforementioned practices act twofold: as a learning tool and also improving the quality of life of the participants and thus, their self-image. All these pioneer experiences were the first step in a process of developing new attitudes contributing the 2030 Agenda for Sustainable Development of United Nations, where among the 17 goals proposed an effective social inclusion of people with disabilities is required. For this, real inclusive practices in geological outreach are imperious. A close cooperation with all the parts (researchers and participants), in a kind of coworking attitude is needed. This Inclusive-Coworking is considered in the sense of social gathering in order to share equal values and look for the synergy that this different outlook implies. And what is more important: the change of role of the previously learners into an active part of the scientific outreach, providing the adequate methodology for that. The offer of non-formal learning activities normally includes the participation of university professors and researchers in Science Week editions. During the 2016 session in Madrid, four adults with intellectual disability who were participants in the previous edition, contributed in the palaeontological workshop. They were in charge of four of the eight modules explaining the origin of fossils and how to collect them, the evolution of equids' limbs, and the main dentition types in vertebrates to the twenty 16 year old secondary students who attended the workshop. During the development of the experience all the students were pleased with the inclusive approach, and the interaction of all participants was fruitful. Although the explanations took a bit more time when made by our functional diverse fellows, all the abstracts concepts

What Counts as Inclusion?

ERIC Educational Resources Information Center

Walton, E.; Nel, N.

2012-01-01

In the years since the publication in South Africa of White Paper Six: Special needs education (Department of Education (DoE) 2001) various schools in the state and independent sectors have begun to implement inclusive policies and practices. With reference to the Guidelines for full-service/inclusive schools issued in 2009, and by discussing a…

The Inclusion Facilitator's Guide

ERIC Educational Resources Information Center

Jorgensen, Cheryl M.; Schuh, Mary C.; Nisbet, Jan

2005-01-01

Inclusion facilitators are educators who do more than teach children with disabilities--they advocate for change in schools and communities, sparking a passion for inclusion in teachers, administrators, and families and giving them the practical guidance they need to make it work. This is an essential new role in today's schools, and this guide…

Understanding Inclusion in Cyprus

ERIC Educational Resources Information Center

Mamas, Christoforos

2013-01-01

This paper provides a framework for understanding inclusion in Cyprus. The evidence base is the result of a six-month qualitative research study in five Cypriot mainstream primary schools. Despite the rhetoric in favour of inclusion, it seems that the Cypriot educational system is still highly segregating in its philosophy and does not fully…

Optimization of inclusive fitness.

PubMed

Grafen, Alan

2006-02-07

The first fully explicit argument is given that broadly supports a widespread belief among whole-organism biologists that natural selection tends to lead to organisms acting as if maximizing their inclusive fitness. The use of optimization programs permits a clear statement of what this belief should be understood to mean, in contradistinction to the common mathematical presumption that it should be formalized as some kind of Lyapunov or even potential function. The argument reveals new details and uncovers latent assumptions. A very general genetic architecture is allowed, and there is arbitrary uncertainty. However, frequency dependence of fitnesses is not permitted. The logic of inclusive fitness immediately draws together various kinds of intra-genomic conflict, and the concept of 'p-family' is introduced. Inclusive fitness is thus incorporated into the formal Darwinism project, which aims to link the mathematics of motion (difference and differential equations) used to describe gene frequency trajectories with the mathematics of optimization used to describe purpose and design. Important questions remain to be answered in the fundamental theory of inclusive fitness.

When social inclusion is not enough: Implicit expectations of extreme inclusion in borderline personality disorder.

PubMed

De Panfilis, Chiara; Riva, Paolo; Preti, Emanuele; Cabrino, Chiara; Marchesi, Carlo

2015-10-01

Increasing evidence suggests that individuals with borderline personality disorder (BPD) might feel rejected even when socially included by others. A psychological mechanism accounting for this response bias could be that objective social inclusion violates BPD patients' underlying implicit needs of "extreme" inclusion. Thus, this study investigated whether, during interpersonal exchanges, BPD patients report more rejection-related negative emotions and less feelings of social connection than controls unless they are faced with conditions of extreme social inclusion. Sixty-one BPD patients and 61 healthy controls completed a modified Cyberball paradigm. They were randomly assigned to a condition of ostracism, social inclusion, or overinclusion (a proxy for extreme social inclusion). They then rated their emotional states and feelings of social connection immediately and 20 min after the game. BPD patients reported greater levels of negative emotions than controls in the ostracism and the inclusion conditions, but not when overincluded. Furthermore, only for BPD participants was overinclusion associated with experiencing less negative emotions than the ostracism condition. However, BPD patients reported lower feelings of social connection than controls in any experimental situation. Thus, in BPD, a laboratory condition of "overinclusion" is associated with a reduction of negative emotions to levels comparable to those of control participants, but not with similar degrees of social connection. These results suggest that for BPD patients, even "including contexts" activate feelings of rejection. Their implicit expectations of idealized interpersonal inclusion may nullify the opportunity of experiencing "real" social connection and explain their distorted subjective experiences of rejection. (c) 2015 APA, all rights reserved).

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

Inclusion 101: How To Teach All Learners.

ERIC Educational Resources Information Center

Bauer, Anne M.; Shea, Thomas M.

This book is designed to help educators provide effective instruction to students with disabilities in inclusive classrooms. Chapters address: (1) the concepts of inclusive society, schools, classrooms and services; (2) legal foundations for inclusion and government support for education; (3) the qualities of inclusive schools and classrooms; (4)…

The Inclusion of Inclusive Education in International Development: Lessons from Papua New Guinea

ERIC Educational Resources Information Center

Le Fanu, Guy

2013-01-01

A new "inclusive" curriculum has been introduced in Papua New Guinea, with significant levels of support from a bilateral development agency. The curriculum is inclusive in the sense that it is designed to meet the diverse, complex, and ever-changing needs of students. Research indicates the curriculum has been shaped by various…

The Inclusion Puzzle: A Case Study of Inclusion in a Rural Elementary School

ERIC Educational Resources Information Center

Arnold, Linda N.

2010-01-01

Inclusion of special education students in general education classrooms has come to general acceptance by educators as one option in the continuum of special education service delivery. Another view of inclusion is the ideal of providing for all the varied individual needs of a diverse population of students: learning needs, physical needs,…

Inclusion evolution in molten and solidifying steel

NASA Astrophysics Data System (ADS)

Wang, Yan

Cleanliness, with respect to impurities and nonmetallic inclusions in the melt, is an important issue in steel production. The commercial interest in controlling steel cleanliness has been growing rapidly, because clean steel exhibits a highly attractive combination of corrosion resistance, good formability, pleasing appearance, and a wide range of strength levels. In order to satisfy the requirements for the degree of cleanliness in steel, controlling the size distribution, chemistry and shape of inclusions are of great importance in the steelmaking process. A knowledge of the formation of nonmetallic inclusions and their chemical and morphological evolution during the steelmaking and casting process is necessary in order to minimize the inclusion size and also try to promote potentially beneficial properties of inclusions, such as grain-refining. In this research, the evolution of inclusions in molten and solidifying steels was investigated through in-situ observations using a high temperature Confocal Scanning Laser Microscope (CSLM). The study focused on solid Al2O3 and liquid Al 2O3-CaO inclusions on low carbon steel melt surfaces. Firstly, the agglomeration and clustering of inclusions on steel surfaces were quantified and compared to predictions according to capillary depression driven attraction forces. A strong agglomeration was observed between the solid Al2O33 particle pairs. However, the liquid Al 2O3-CaO inclusions were not prone to agglomeration due to their lens-like morphology, which causes the absence of capillary force. Secondly, the pushing vs. engulfment and entrapment of both liquid Al 2O3-CaO and solid Al2O3 inclusions by advancing planar and cellular delta-ferrite solidification fronts was studied and compared to model predictions based on the force balances acting on the inclusions at the solid/melt interface. The critical velocity, above which the inclusions get engulfed, was observed to be slower at the cellular front than at the planar

Utilizing the Critical Inclusive Praxis: The Voyage of Five Selected School Principals in Building Inclusive School Cultures

ERIC Educational Resources Information Center

Osiname, Ayodeji Tolulope

2018-01-01

This article integrates relevant literature with the lived experiences of five school principals regarding how they utilized different leadership styles to build an inclusive school culture. The conceptual framework--a Critical Inclusive Praxis, including culture, change, leadership, inclusion and challenge--provided a base for the literature and…

Supporting the Transition to Inclusive Education: Teachers' Attitudes to Inclusion in the Seychelles

ERIC Educational Resources Information Center

Main, Susan; Chambers, Dianne J.; Sarah, Paulette

2016-01-01

The inclusion of children with disability in regular classroom settings has been identified worldwide as crucial to the provision of effective education for all children and to the creation of more inclusive societies. To this end there has been significant focus on pre-service and in-service teacher education to ensure that teachers are…

Neuronal intranuclear inclusions are ultrastructurally and immunologically distinct from cytoplasmic inclusions of neuronal intermediate filament inclusion disease

PubMed Central

Mosaheb, Sabrina; Thorpe, Julian R.; Hashemzadeh-Bonehi, Lida; Bigio, Eileen H.; Gearing, Marla; Cairns, Nigel J.

2006-01-01

Abnormal neuronal cytoplasmic inclusions (NCIs) containing aggregates of α-internexin and the neurofilament (NF) subunits, NF-H, NF-M, and NF-L, are the signature lesions of neuronal intermediate filament (IF) inclusion disease (NIFID). The disease has a clinically heterogeneous phenotype, including fronto-temporal dementia, pyramidal and extrapyramidal signs presenting at a young age. NCIs are variably ubiquitinated and about half of cases also have neuronal intranuclear inclusions (NIIs), which are also ubiquitinated. NIIs have been described in polyglutamine-repeat expansion diseases, where they are strongly ubiquitin immunoreactive. The fine structure of NIIs of NIFID has not previously been described. Therefore, to determine the ultrastructure of NIIs, immunoelectron microscopy was undertaken on NIFID cases and normal aged control brains. Our results indicate that the NIIs of NIFID are strongly ubiquitin immunoreactive. However, unlike NCIs which contain ubiquitin, α-internexin and NF epitopes, NIIs contain neither epitopes of α-internexin nor NF subunits. Neither NIIs nor NCIs were recognised by antibodies to expanded polyglutamine repeats. The NII of NIFID lacks a limiting membrane and contains straight filaments of 20 nm mean width (range 11–35 nm), while NCIs contain filaments with a mean width of 10 nm (range 5–18 nm; t-test, P<0.001). Biochemistry revealed no differences in neuronal IF protein mobilities between NIFID and normal brain tissue. Therefore, NIIs of NIFID contain filaments morphologically and immunologically distinct from those of NCIs, and both types of inclusion lack expanded polyglutamine tracts of the triplet-repeat expansion diseases. These observations indicate that abnormal protein aggregation follows separate pathways in different neuronal compartments of NIFID. PMID:16025283

Inclusive Attitudes: A Pre-Service Analysis

ERIC Educational Resources Information Center

Ryan, Thomas G.

2009-01-01

This enquiry has shown that pre-service teachers in Ontario, Canada (n = 141), believe they have the prerequisite training and knowledge to be inclusive educators. They believe inclusion is effective and produces required results for both identified and non-identified students. Inclusion was viewed as a difficult proposition dependent upon several…

Inclusion: By Choice or by Chance?

ERIC Educational Resources Information Center

Armstrong, Derrick; Armstrong, Ann Cheryl; Spandagou, Ilektra

2011-01-01

This paper explores the development internationally of the inclusive education perspective. Inclusive education as a late modernity reform project is exemplified in the call for "Education for All". Despite the simplicity of its message, inclusion is highly contestable. We argue in this paper that the key questions raised by the concept of…

Melatonin mitigates cadmium phytotoxicity through modulation of phytochelatins biosynthesis, vacuolar sequestration, and antioxidant potential in Solanum lycopersicum L

PubMed Central

Hasan, Md. Kamrul; Ahammed, Golam Jalal; Yin, Lingling; Shi, Kai; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Zhou, Jie

2015-01-01

Melatonin is a ubiquitous signal molecule, playing crucial roles in plant growth and stress tolerance. Recently, toxic metal cadmium (Cd) has been reported to regulate melatonin content in rice; however, the function of melatonin under Cd stress, particularly in higher plants, still remains elusive. Here, we show that optimal dose of melatonin could effectively ameliorate Cd-induced phytotoxicity in tomato. The contents of Cd and melatonin were gradually increased over time under Cd stress. However, such increase in endogenous melatonin was incapable to reverse detrimental effects of Cd. Meanwhile, supplementation with melatonin conferred Cd tolerance as evident by plant biomass and photosynthesis. In addition to notable increase in antioxidant enzymes activity, melatonin-induced Cd stress mitigation was closely associated with enhanced H+-ATPase activity and the contents of glutathione and phytochelatins. Although exogenous melatonin had no effect on root Cd content, it significantly reduced leaf Cd content, indicating its role in Cd transport. Analysis of Cd in different subcellular compartments revealed that melatonin increased cell wall and vacuolar fractions of Cd. Our results suggest that melatonin-induced enhancements in antioxidant potential, phytochelatins biosynthesis and subsequent Cd sequestration might play a critical role in plant tolerance to Cd. Such a mechanism may have potential implication in safe food production. PMID:26322055

Towards inclusive occupational therapy: Introducing the CORE approach for inclusive and occupation-focused practice.

PubMed

Pereira, Robert B

2017-12-01

Occupation is a human right and a social determinant of health. It is also taken for granted. Having access to, and participating in, occupation, is intricately linked to positive health and wellbeing. Despite theory and evidence to support the link between occupation, health and wellbeing, occupational therapists can struggle with applying an occupation focus in practice and knowing how to use occupational frameworks to enable occupation. The purpose of this paper is to introduce the Capabilities, Opportunities, Resources and Environments (CORE) approach for inclusive and occupation-focused practice. It provides occupational therapists with a means of operationalising occupational enablement and facilitating social inclusion. The CORE approach is introduced by linking its main ideas to Economist and Nobel Prize Laureate Amartya Sen's capabilities approach, as well as findings from the author's doctoral research into entrenched disadvantage and social inclusion. Practical questions guided by the CORE approach's acronym are given to explore how the approach can be utilised alongside other occupational models and frameworks to encourage strategies for effective enablement through occupation for social inclusion. As experts in enabling occupation, occupational therapists can use the CORE approach to design occupation-focused interventions and promote inclusive occupational therapy. © 2017 Occupational Therapy Australia.

Saudi Children's Thoughts on Inclusive Education

ERIC Educational Resources Information Center

Dare, Lynn; Nowicki, Elizabeth; Felimban, Huda

2017-01-01

Saudi Arabia has followed the global trend towards inclusive education; however, few researchers have examined the lived reality of inclusion in Saudi schools. In this study, we interviewed 31 girls and 34 boys in grades 5 and 6 who attended an inclusive school in Saudi. The overarching research question was, "How do Saudi children perceive…

The added value of inclusive research.

PubMed

Walmsley, Jan; Strnadová, Iva; Johnson, Kelley

2017-12-12

The study asks when does inclusive research add value? The authors argue that this is important, given the additional time and cost of co-researching with people with intellectual disabilities. The study is situated in debates about a "second generation" of inclusive research which advocates focussing more on outcomes than process. The authors argue that this is premature, rather the authors propose that inclusive research is valuable when it helps to recognize, foster, and above all communicate the contributions people with intellectual disabilities can make. The authors conducted a literature review of 52 peer-reviewed journal articles about inclusive research and analysed them.. The authors conclude that inclusive research adds value when there is a distinctive contribution which only co-researchers with intellectual disabilities can make, when it highlights the contributions people with intellectual disabilities make, and when it contributes to better lives for the wider population of people with intellectual disabilities. The authors propose a revised definition of inclusive research to replace that published by Walmsley and Johnson in 2003. © 2017 John Wiley & Sons Ltd.

Leadership for Inclusion: Conceptualising and Enacting Inclusion in Integrated Schools in a Troubled Society

ERIC Educational Resources Information Center

McGlynn, Claire; London, Tim

2013-01-01

Inclusion is increasingly understood as an educational reform that responds to the diversity of all learners, challenging the marginalisation, exclusion and underachievement which may result from all forms of "difference". Leadership for inclusion is conceptualised here as driving a constant struggle to create shared meanings of…

Protein Mobilization in Germinating Mung Bean Seeds Involves Vacuolar Sorting Receptors and Multivesicular Bodies1[W][OA

PubMed Central

Wang, Junqi; Li, Yubing; Lo, Sze Wan; Hillmer, Stefan; Sun, Samuel S.M.; Robinson, David G.; Jiang, Liwen

2007-01-01

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination. PMID:17322331

Axonal inclusions in the crab Hemigrapsus nudus.

PubMed

Smith, R S

1978-10-01

Light microscopic examination of living giant axons from the walking legs of Hemigrapsus nudus revealed intra-axonal inclusions which were usually several tens of micrometers long and about 5 micron wide. The inclusions were filled with small light-scattering particles. The inclusions were shown, by thin section electron microscopy, to be composed largely 68% by volume) of mitochondria. Each inclusion was surrounded by membrane bounded spaces which are presumed to represent a part of the smooth endoplasmic reticulum. Similar inclusions were not found in the leg axons of a variety of other decapod crustaceans.

Social inclusion and mental health.

PubMed

Cobigo, Virginie; Stuart, Heather

2010-09-01

Recent research on approaches to improving social inclusion for people with mental disabilities is reviewed. We describe four approaches (or tools) that can be used to improve social inclusion for people with mental disabilities: legislation, community-based supports and services, antistigma/antidiscrimination initiatives, and system monitoring and evaluation. While legislative solutions are the most prevalent, and provide an important framework to support social inclusion, research shows that their full implementation remains problematic. Community-based supports and services that are person-centered and recovery-oriented hold considerable promise, but they are not widely available nor have they been widely evaluated. Antistigma and antidiscrimination strategies are gaining in popularity and offer important avenues for eliminating social barriers and promoting adequate and equitable access to care. Finally, in the context of the current human rights and evidence-based health paradigms, systematic evidence will be needed to support efforts to promote social inclusion for people with mental disabilities, highlight social inequities, and develop best practice approaches. Tools that promote social inclusion of persons with mental disabilities are available, though not yet implemented in a way to fully realize the goals of current disability discourse.

Transmit: An Advanced Traffic Management System

DOT National Transportation Integrated Search

1995-11-27

TRANSCOM'S SYSTEM FOR MANAGING INCIDENTS AND TRAFFIC, KNOWN AS TRANSMIT, WAS INITIATED TO ESTABLISH THE FEASIBILITY OF USING AUTOMATIC VEHICLE IDENTIFICATION (AVI) EQUIPMENT FOR TRAFFIC MANAGEMENT AND SURVEILLANCE APPLICATIONS. AVI TECHNOLOGY SYSTEMS...

Inclusion body myositis.

PubMed

Dimachkie, Mazen M; Barohn, Richard J

2012-07-01

The idiopathic inflammatory myopathies are a group of rare disorders that share many similarities. These include dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM), and sporadic inclusion body myositis (IBM). Inclusion body myositis is the most common idiopathic inflammatory myopathy after age 50 and it presents with chronic proximal leg and distal arm asymmetric mucle weakness. Despite similarities with PM, it is likely that IBM is primarily a degenerative disorder rather than an inflammatory muscle disease. Inclusion body myositis is associated with a modest degree of creatine kinase (CK) elevation and an abnormal electromyogram demonstrating an irritative myopathy with some chronicity. The muscle histopathology demonstrates inflammatory exudates surrounding and invading nonnecrotic muscle fibers often times accompanied by rimmed vacuoles. In this chapter, we review sporadic IBM. We also examine past, essentially negative, clinical trials in IBM and review ongoing clinical trials. For further details on DM, PM, and NM, the reader is referred to the idiopathic inflammatory myopathies chapter. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Suddenly included: cultural differences in experiencing re-inclusion.

PubMed

Pfundmair, Michaela; Graupmann, Verena; Du, Hongfei; Frey, Dieter; Aydin, Nilüfer

2015-03-01

In the current research, we examined whether re-inclusion (i.e. the change from a previous state of exclusion to a new state of inclusion) was perceived differently by people with individualistic and collectivistic cultural backgrounds. Individualists (German and Austrian participants) but not collectivists (Chinese participants) experienced re-inclusion differently than continued inclusion: While collectivistic participants did not differentiate between both kinds of inclusion, individualistic participants showed reduced fulfilment of their psychological needs under re-inclusion compared to continued inclusion. The results moreover revealed that only participants from individualistic cultures expressed more feelings of exclusion when re-included than when continually included. These exclusionary feelings partially mediated the relationship between the different states of inclusion and basic need fulfilment. © 2014 International Union of Psychological Science.

Periodic Inclusion—Matrix Microstructures with Constant Field Inclusions

NASA Astrophysics Data System (ADS)

Liu, Liping; James, Richard D.; Leo, Perry H.

2007-04-01

We find a class of special microstructures consisting of a periodic array of inclusions, with the special property that constant magnetization (or eigenstrain) of the inclusion implies constant magnetic field (or strain) in the inclusion. The resulting inclusions, which we term E-inclusions, have the same property in a finite periodic domain as ellipsoids have in infinite space. The E-inclusions are found by mapping the magnetostatic or elasticity equations to a constrained minimization problem known as a free-boundary obstacle problem. By solving this minimization problem, we can construct families of E-inclusions with any prescribed volume fraction between zero and one. In two dimensions, our results coincide with the microstructures first introduced by Vigdergauz,[1,2] while in three dimensions, we introduce a numerical method to calculate E-inclusions. E-inclusions extend the important role of ellipsoids in calculations concerning phase transformations and composite materials.

Numerical Simulations of Inclusion Behavior in Gas-Stirred Ladles

NASA Astrophysics Data System (ADS)

Lou, Wentao; Zhu, Miaoyong

2013-06-01

A computation fluid dynamics-population balance model (CFD-PBM) coupled model has been proposed to investigate the bubbly plume flow and inclusion behavior including growth, size distribution, and removal in gas-stirred ladles, and some new and important phenomena and mechanisms were presented. For the bubbly plume flow, a modified k- ɛ model with extra source terms to account for the bubble-induced turbulence was adopted to model the turbulence, and the bubble turbulent dispersion force was taken into account to predict gas volume fraction distribution in the turbulent gas-stirred system. For inclusion behavior, the phenomena of inclusions turbulent random motion, bubbles wake, and slag eye forming on the molten steel surface were considered. In addition, the multiple mechanisms both that promote inclusion growth due to inclusion-inclusion collision caused by turbulent random motion, shear rate in turbulent eddy, and difference inclusion Stokes velocities, and the mechanisms that promote inclusion removal due to bubble-inclusion turbulence random collision, bubble-inclusion turbulent shear collision, bubble-inclusion buoyancy collision, inclusion own floatation near slag-metal interface, bubble wake capture, and wall adhesion were investigated. The importance of different mechanisms and total inclusion removal ratio under different conditions, and the distribution of inclusion number densities in ladle, were discussed and clarified. The results show that at a low gas flow rate, the inclusion growth is mainly attributed to both turbulent shear collision and Stokes collision, which is notably affected by the Stokes collision efficiency, and the inclusion removal is mainly attributed to the bubble-inclusion buoyancy collision and inclusion own floatation near slag-metal interface. At a higher gas flow rate, the inclusions appear as turbulence random motion in bubbly plume zone, and both the inclusion-inclusion and inclusion-bubble turbulent random collisions become

The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

PubMed

Miles, Anna L; Burr, Stephen P; Grice, Guinevere L; Nathan, James A

2017-03-15

Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

Beliefs in social inclusion: Invariance in associations among hope, dysfunctional attitudes, and social inclusion across adolescence and young adulthood.

PubMed

Berry, Clio; Greenwood, Kathryn

2017-09-19

Social disability in youth is an important precursor of long-term social and mental health problems. Social inclusion is a key policy driver and fits well within a new paradigm of health and well-being rather than illness-oriented services, yet little is known about social inclusion and its facilitators for "healthy" young people. We present a novel exploratory structural analysis of social inclusion using measures from 387 14- to 36-year-olds. Our model represents social inclusion as comprising social activity and community belonging, with both domains predicted by hopeful and dysfunctional self-beliefs but hopefulness more uniquely predicting social inclusion in adolescence. We conclude that social inclusion can be modeled for meaningful comparison across spectra of development, mental health, and functioning.

Indonesian teachers' epistemological beliefs and inclusive education.

PubMed

Sheehy, Kieron; Budiyanto; Kaye, Helen; Rofiah, Khofidotur

2017-01-01

A growing number of children with intellectual disabilities attend inclusive schools in Indonesia. Previous research has suggested that teachers' type of school and experience influences their beliefs about inclusive education. This research collected questionnaire data from 267 Indonesian teachers and compared the responses from those working in inclusive, special and regular schools regarding their epistemological and pedagogical beliefs. The results showed that teachers in inclusive schools expressed stronger social constructivist beliefs than those in other schools. However, it was teachers' epistemological beliefs, rather than their type of school or experience, which were the significant predictor of their beliefs about inclusive education. The findings suggest that international epistemological research needs to have a more nuanced view of constructivist models of learning to better understand and inform how inclusive pedagogy is being enacted in different contexts.

The Inclusive Classroom. Professional's Guide.

ERIC Educational Resources Information Center

Grenot-Scheyer, Marquita; And Others

Inclusive education reflects the changing culture of contemporary schools with emphasis on active learning, authentic assessment practices, applied curriculum, multi-level instructional approaches, and increased attention to diverse student needs and individualization. This guide is intended to help teachers implement inclusive educational…

Building Inclusion from the Ground up: A Review of Whole School Re-Culturing Programmes for Sustaining Inclusive Change

ERIC Educational Resources Information Center

McMaster, Christopher

2013-01-01

This paper suggests that whole school re-culturing programmes can potentially assist in the creation of more inclusive value orientated schools. The relationship between school culture and successful inclusion has been demonstrated in the literature. Furthermore, the structure of whole school programmes in inculcating inclusive values and…

Measuring Attitudes toward Inclusive Education.

ERIC Educational Resources Information Center

Wilczenski, Felicia L.

1992-01-01

Developed scale to measure attitudes toward inclusive education, in which disabled students are responsibility of regular teacher supported by specialists. Administered scale to 301 elementary and secondary teachers and to 144 undergraduate elementary education majors. Analysis yielded four discrete dimensions of inclusive education with…

Nuclear Glycogen Inclusions in Canine Parietal Cells.

PubMed

Silvestri, S; Lepri, E; Dall'Aglio, C; Marchesi, M C; Vitellozzi, G

2017-05-01

Nuclear glycogen inclusions occur infrequently in pathologic conditions but also in normal human and animal tissues. Their function or significance is unclear. To the best of the authors' knowledge, no reports of nuclear glycogen inclusions in canine parietal cells exist. After initial observations of nuclear inclusions/pseudoinclusions during routine histopathology, the authors retrospectively examined samples of gastric mucosa from dogs presenting with gastrointestinal signs for the presence of intranuclear inclusions/pseudoinclusions and determined their composition using histologic and electron-microscopic methods. In 24 of 108 cases (22%), the authors observed various numbers of intranuclear inclusions/pseudoinclusions within scattered parietal cells. Nuclei were characterized by marked karyomegaly and chromatin margination around a central optically empty or slightly eosinophilic area. The intranuclear inclusions/pseudoinclusions stained positive with periodic acid-Schiff (PAS) and were diastase sensitive, consistent with glycogen. Several PAS-positive/diastase-sensitive sections were further examined by transmission electron microscopy, also using periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining to identify polysaccharides. Ultrastructurally, the nuclear inclusions were composed of electron-dense particles that were not membrane bound, without evidence of nuclear membrane invaginations or cytoplasmic organelles in the nuclei, and positive staining with PA-TCH-SP, confirming a glycogen composition. No cytoplasmic glycogen deposits were observed, suggesting that the intranuclear glycogen inclusions were probably synthesized in loco. Nuclear glycogen inclusions were not associated with gastritis or colonization by Helicobacter-like organisms ( P > .05). Our findings suggest that nuclear glycogen inclusions in canine parietal cells could be an incidental finding. Nevertheless, since nuclear glycogen is present in several pathologic

75 FR 1289 - Minority and Women Inclusion

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-11

... ensure the inclusion of minorities, women, and individuals with disabilities, and businesses owned by... inclusion and utilization of minorities, women, individuals with disabilities, and minority-, women-, and... the inclusion and utilization of minorities, women, individuals with disabilities, and minority...

The Evolution of Secondary Inclusion.

ERIC Educational Resources Information Center

Thousand, Jacqueline; Rosenberg, Richard L.; Bishop, Kathryn D.; Villa, Richard A.

1997-01-01

Offers an alternative "Circle of Courage" model of education, derived from Native American culture, for creating inclusive high schools that welcome, value, support, and facilitate the learning of adolescents with differing abilities. Best practices related to curriculum, instruction, assessment, and campus life for effective inclusion are…

Preschool Inclusion Finance Toolkit 2017

ERIC Educational Resources Information Center

Cate, Debbie; Peters, Mary

2017-01-01

This document outlines strategies and guidance on collaborative funding strategies and mechanisms, including braided funding, to create and sustain high-quality inclusive regular early childhood settings that are inclusive of preschool children with disabilities. The use of federal funds is outlined specific to supporting young children with…

Structure and organization of nanosized-inclusion-containing bilayer membranes

NASA Astrophysics Data System (ADS)

Ren, Chun-Lai; Ma, Yu-Qiang

2009-07-01

Based on a considerable amount of experimental evidence for lateral organization of lipid membranes which share astonishingly similar features in the presence of different inclusions, we use a hybrid self-consistent field theory (SCFT)/density-functional theory (DFT) approach to deal with bilayer membranes embedded by nanosized inclusions and explain experimental findings. Here, the hydrophobic inclusions are simple models of hydrophobic drugs or other nanoparticles for biomedical applications. It is found that lipid/inclusion-rich domains are formed at moderate inclusion concentrations and disappear with the increase in the concentration of inclusions. At high inclusion content, chaining of inclusions occurs due to the effective depletion attraction between inclusions mediated by lipids. Meanwhile, the increase in the concentration of inclusions can also cause thickening of the membrane and the distribution of inclusions undergoes a layering transition from one-layer structure located in the bilayer midplane to two-layer structure arranged into the two leaflets of a bilayer. Our theoretical predictions address the complex interactions between membranes and inclusions suggesting a unifying mechanism which reflects the competition between the conformational entropy of lipids favoring the formation of lipid- and inclusion-rich domains in lipids and the steric repulsion of inclusions leading to the uniform dispersion.

Struggling for inclusive education in the North and the South: educators' perceptions on inclusive education in Finland and Zambia.

PubMed

Moberg, Sakari; Savolainen, Hannu

2003-03-01

A survey assessed the perceptions of 1350 Zambian teachers and parents and 512 Finnish teachers regarding inclusive education and consequently the best placement for children with different disabilities. On the whole, perceptions varied but were quite critical. On inclusion in general, the regular (also termed "ordinary") Finnish teachers were the most critical group and the Finnish special education teachers the most optimistic. Most respondents felt that inclusive education enhances social justice. However, the pursuit of inclusion in practice, especially the guarantee of good and effective education for all, was seen as problematic. Compared with Finnish respondents, the Zambian respondents preferred a more segregated educational environment for children with different disabilities. Type and severity of disability affected the preferred educational setting and there were clear differences in this regard between the respondents from the two countries. The findings support the idea that educators' attitudes towards inclusion are important in developing inclusive school systems and that inclusive education is best understood as a multidimensional concept that, at the practical level, is highly context-dependent.

Silicate Inclusions in IAB Irons: Correlations Between Metal Composition and Inclusion Properties, and Inferences for Their Origin

NASA Astrophysics Data System (ADS)

Benedix, G. K.; McCoy, T. J.; Keil, K.

1995-09-01

IAB irons are the largest group of iron meteorites, exhibit a large range of siderophile element concentrations in their metal, and commonly contain silicate inclusions with roughly chondritic composition. They are closely related to IIICD irons [1,2] and their inclusions resemble winonaites [3]. It has been suggested that IAB's and IIICD's formed in individual impact melt pools [4,2] on a common parent body. However, it has also been suggested that fractional crystallization [5,6] of a S-saturated core could produce the observed siderophile element trends. Metal composition is correlated with silicate inclusion mineralogy in IIICD's [1], indicating reactions between solid silicates and the metallic magma in a core. These trends observed in IIICD's differ from those in IAB's, suggesting different parent bodies. A bi-modal grouping, based primarily on mineralogy and mineral abundances, was suggested for IAB inclusions [7]. However, recent recoveries of several new silicate-bearing IAB's, along with the emergence of new ideas on their origins, prompted a comprehensive study to document more fully the range of inclusions within IAB irons, to examine possible correlations between the compositions of the metallic host and the silicate inclusions, and to elucidate the origin of IAB irons. We are studying troilite-graphite-silicate inclusions in 24 IAB irons with Ni concentrations ranging from 6.6-25.0%. These include Odessa and Copiapo types [7], newly recovered meteorites (e.g., Lueders [8]) and meteorites with extreme Ni contents (e.g., Jenny's Creek, 6.8%; San Cristobal, 25.0% [9]). The inclusions exhibit a range of textures from recrystallized to partial melts (e.g., Caddo County [10]). Rigorous classification [7] is hampered by heterogeneities between group meteorites, between different samples of distinct meteorites, and within individual inclusions. While intergroup heterogeneities make comparisons between the suite of IAB's somewhat difficult, some general trends

Interaction between a crack and a soft inclusion

NASA Technical Reports Server (NTRS)

Xue-Hui, L.; Erdogan, F.

1985-01-01

With the application to weld defects in mind, the interaction problem between a planar-crack and a flat inclusion in an elastic solid is considered. The elastic inclusion is assumed to be sufficiently thin so that the thickness distribution of the stresses in the inclusion may be neglected. The problem is reduced to a system of four integral equations having Cauchy-type dominant kernels. The stress intensity factors are calculated and tabulated for various crack-inclusion geometries and the inclusion to matrix modulus ratios, and for general homogeneous loadiong conditions away from the crack-inclusion region.

Human Signatures for Personnel Detection

DTIC Science & Technology

2010-09-14

work\\ TESIS \\prueba.avi’); switch videoinfo.ImageType case ’truecolor’ video=aviread(’C:\\MATLAB\\R2006a\\work\\ TESIS \\prueba.avi’); case...8217indexed’ video=aviread(’C:\\MATLAB\\R2006a\\work\\ TESIS \\prueba.avi’); video=ind2rgb(video); end 4.1.2 Color to Grayscale Converter The conversion from...algorithm. • Maximum errors for both GTSig and the lattice Boltzmann method were confined to the corners where fthe temperature is ill de ined Numerical

IDEA and Early Childhood Inclusion.

ERIC Educational Resources Information Center

Smith, Barbara J.; Rapport, Mary Jane K.

This paper discusses 1997 amendments to the Individuals with Disabilities Education Act (IDEA) that promote the inclusion of children with disabilities in general early childhood education settings. The evolution of inclusion policy is explored and changes in disability terminology are described. Amended provisions are then explained and include:…

Early Childhood Inclusion in Turkey

ERIC Educational Resources Information Center

Diken, Ibrahim H.; Rakap, Salih; Diken, Ozlem; Tomris, Gozde; Celik, Secil

2016-01-01

Inclusion of young children with disabilities into regular preschool classrooms is a common practice that has been implemented for several decades in industrialized nations around the world, and many developing countries including Turkey have been developing and implementing laws, regulation, and services to support inclusion and teaching in…

Inclusive Education in Georgia: Current Trends and Challenges

ERIC Educational Resources Information Center

Tchintcharauli, Tinatin; Javakhishvili, Nino

2017-01-01

This article presents the first comprehensive study of the Georgian inclusive education system launched nationwide in 2006. An internationally recognised tool, the "Pathway to Inclusion--Barometer of Inclusive Education", was applied within the country to highlight the current position of inclusive education in Georgia. The collected…

Characteristics of Inclusive Faith Communities: A Preliminary Survey of Inclusive Practices in the United States

ERIC Educational Resources Information Center

Griffin, Megan M.; Kane, Lydia W.; Taylor, Courtney; Francis, Susan H.; Hodapp, Robert M.

2012-01-01

Background: Although participation in faith communities is important to many individuals with disabilities, few studies have examined differences between communities that are more (versus less) inclusive. This study investigated characteristics of faith communities in the United States related to greater inclusion. Methods: Participants were 160…

Teaching Inclusively: Are Secondary Physical Education Student Teachers Sufficiently Prepared to Teach in Inclusive Environments?

ERIC Educational Resources Information Center

Coates, Janine Kim

2012-01-01

Background: Contemporary British educational guidelines, such as the National Curriculum (NC) have adopted inclusivity in the way children with special educational needs (SEN) are taught. Therefore, inclusion has risen up the political agenda, resulting in more children with SEN being taught in mainstream environments. Empirical research has…

The Inclusion of Music/the Music of Inclusion

ERIC Educational Resources Information Center

Lubet, Alex

2009-01-01

The intention of this paper is to situate music within inclusive education. Intersections of music--widely regarded as a "talent" or hyperability--and disability provide unique perspectives on social organisation in general and human valuation in particular. Music is a ubiquitous and an essential component of learning beginning in infancy.…

Feature Issue on Inclusion and School Restructuring.

ERIC Educational Resources Information Center

Vandercook, Terri; York-Barr, Jennifer

1996-01-01

This feature issue on inclusion and school restructuring for students with and without disabilities contains the following articles: (1) "The 7 Habits for Educators" (Terri Vandercook); (2) "The Evolution of Inclusive Education" (Jennifer York-Barr and Terri Vandercook); (3) "Creating Inclusive Schools: What Does the…

Forging Inclusive Solutions: Experiential Earth Charter Education

ERIC Educational Resources Information Center

Hill, Linda D.

2010-01-01

Forging Inclusive Solutions describes the aims, methodology and outcomes of Inclusive Leadership Adventures, an experiential education curriculum for exploring the Earth Charter. Experiential education builds meaningful relationships, skills, awareness and an inclusive community based on the Earth Charter principles. When we meet people where they…

Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines

PubMed Central

Chandel, Avinash; Das, Krishna K.; Bachhawat, Anand K.

2016-01-01

Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeast. We show here that glutathione depletion in yeast leads to the activation of two cytoplasmically inward-facing channels: the plasma membrane, Cch1p, and the vacuolar calcium channel, Yvc1p. Deletion of these channels partially rescues cells from glutathione depletion–induced cell death. Subsequent investigations on the Yvc1p channel, a homologue of the mammalian TRP channels, revealed that the channel is activated by glutathionylation. Yvc1p has nine cysteine residues, of which eight are located in the cytoplasmic regions and one on the transmembrane domain. We show that three of these cysteines, Cys-17, Cys-79, and Cys-191, are specifically glutathionylated. Mutation of these cysteines to alanine leads to a loss in glutathionylation and a concomitant loss in calcium channel activity. We further investigated the mechanism of glutathionylation and demonstrate a role for the yeast glutathione S-transferase Gtt1p in glutathionylation. Yvc1p is also deglutathionylated, and this was found to be mediated by the yeast thioredoxin, Trx2p. A model for redox activation and deactivation of the yeast Yvc1p channel is presented. PMID:27708136

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

Inclusion in Malaysian Integrated Preschools

ERIC Educational Resources Information Center

Sukumaran, Sailajah; Loveridge, Judith; Green, Vanessa A.

2015-01-01

Inclusive education has been introduced through a number of policy developments in Malaysia over the last 10 years but there is little research investigating the extent and nature of inclusive education for preschoolers with special educational needs (SEN). This study surveyed both regular and special education teachers in Malaysian integrated…

Early Childhood Inclusion in Spain

ERIC Educational Resources Information Center

Giné, Climent; Balcells-Balcells, Anna; Cañadas, Margarita; Paniagua, Gema

2016-01-01

This article describes early childhood inclusion in educational settings in Spain. First, we address the legislative framework of preschool education in Spain and offer a brief analysis of some relevant issues, including the current situation of early childhood education and inclusion at this stage. Second, current policies and practices relating…

Child wellness and social inclusion: values for action.

PubMed

Prilleltensky, Isaac

2010-09-01

Participatory Action Research (PAR) with children and youth is at the intersection of child wellness and social inclusion. Exclusion and marginalization detract from personal and collective health. Inclusion, on the contrary, contributes to wellness. Hence, we should study inclusion and exclusion in the overall context of child wellness. This special issue offers a wealth of methodologies and lessons for fostering inclusion of young people through PAR. In an effort to synthesize my concerns with child wellness, inclusion, and the scholarly work of this special issue, this paper will (a) articulate the values underpinning the philosophy of social inclusion and child wellness, (b) suggest roles and responsibilities for putting these values into action, and (c) integrate the contributions of this special issue into the emerging framework for social inclusion and child wellness.

Creating LGBTQ-Inclusive Care and Work Environments.

PubMed

Jones-Schenk, Jan

2018-04-01

In considering the full depth of inclusion in care and work environments (and developing inclusive engagement skills for lesbian, gay, bisexual, transgender, queer or questioning [LGBTQ] patients and their families), professional development leaders must bring these discussions and shared learnings into the open. Understanding the LGBTQ population's unique needs is essential to providing personalized health care, and inclusive work environments help to foster more inclusive care for this population. J Contin Educ Nurs. 2018;49(4):151-153. Copyright 2018, SLACK Incorporated.

75 FR 81395 - Minority and Women Inclusion

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-28

... 2590-AA28 Minority and Women Inclusion AGENCIES: Federal Housing Finance Board; Federal Housing Finance... and the inclusion of women and minorities in all activities. The final rule implements the provisions.... It also requires each regulated entity to establish an Office of Minority and Women Inclusion, or...

75 FR 10446 - Minority and Women Inclusion

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-08

... 2590-AA28 Minority and Women Inclusion AGENCIES: Federal Housing Finance Board; Federal Housing Finance... minority and women inclusion. Section 1116 of the Housing and Economic Recovery Act of 2008 amended section... Loan Banks to promote diversity and the inclusion of women and minorities in all activities...

Spectral CT Image Restoration via an Average Image-Induced Nonlocal Means Filter.

PubMed

Zeng, Dong; Huang, Jing; Zhang, Hua; Bian, Zhaoying; Niu, Shanzhou; Zhang, Zhang; Feng, Qianjin; Chen, Wufan; Ma, Jianhua

2016-05-01

Spectral computed tomography (SCT) images reconstructed by an analytical approach often suffer from a poor signal-to-noise ratio and strong streak artifacts when sufficient photon counts are not available in SCT imaging. In reducing noise-induced artifacts in SCT images, in this study, we propose an average image-induced nonlocal means (aviNLM) filter for each energy-specific image restoration.  Methods:  The present aviNLM algorithm exploits redundant information in the whole energy domain. Specifically, the proposed aviNLM algorithm yields the restored results by performing a nonlocal weighted average operation on the noisy energy-specific images with the nonlocal weight matrix between the target and prior images, in which the prior image is generated from all of the images reconstructed in each energy bin.  Results: Qualitative and quantitative studies are conducted to evaluate the aviNLM filter by using the data of digital phantom, physical phantom, and clinical patient data acquired from the energy-resolved and -integrated detectors, respectively. Experimental results show that the present aviNLM filter can achieve promising results for SCT image restoration in terms of noise-induced artifact suppression, cross profile, and contrast-to-noise ratio and material decomposition assessment. Conclusion and Significance: The present aviNLM algorithm has useful potential for radiation dose reduction by lowering the mAs in SCT imaging, and it may be useful for some other clinical applications, such as in myocardial perfusion imaging and radiotherapy.

Attempts to identify the source of avian vacuolar myelinopathy for waterbirds

USGS Publications Warehouse

Rocke, T.E.; Thomas, N.J.; Meteyer, C.U.; Quist, C.F.; Fischer, John R.; Augspurger, T.; Ward, S.E.

2005-01-01

Attempts were made to reproduce avian vacuolar myelinopathy (AVM) in a number of test animals in order to determine the source of the causative agent for birds and to find a suitable animal model for future studies. Submerged vegetation, plankton, invertebrates, forage fish, and sediments were collected from three lakes with ongoing outbreaks of AVM and fed to American coots (Fulica americana), mallard ducks and ducklings (Anas platyrhynchos), quail (Coturnix japonica), and laboratory mice either via gavage or ad libitum. Tissues from AVM-affected coots with brain lesions were fed to ducklings, kestrels (Falco sparverius), and American crows (Corvus brachyrhynchos). Two mallards that ingested one sample of Hydrilla verticillata along with any biotic or abiotic material associated with its external surface developed brain lesions consistent with AVM, although neither of the ducks had clinical signs of disease. Ingestion of numerous other samples of Hydrilla from the AVM affected lakes and a lake with no prior history of AVM, other materials (sediments, algae, fish, invertebrates, and water from affected lakes), or tissues from AVM-affected birds did not produce either clinical signs or brain lesions in any of the other test animals in our studies. These results suggest that waterbirds are most likely exposed to the causative agent of AVM while feeding on aquatic vegetation, but we do not believe the vegetation itself is the agent. We hypothesize that the causative agent of AVM might either be accumulated by aquatic vegetation, such as Hydrilla, or associated with biotic or abiotic material on its external surfaces. In support of that hypothesis, two coots that ingested Hydrilla sampled from a lake with an ongoing AVM outbreak in wild birds developed neurologic signs within 9 days (ataxia, limb weakness, and incoordination), and one of two coots that ingested Hydrilla collected from the same site 13 days later became sick and died within 38 days. None of these three

Genesis of diamond inclusions: An integrated cathodoluminescence (CL) and Electron backscatter diffraction (EBSD) study on eclogitic and peridotitic inclusions and their diamond host.

NASA Astrophysics Data System (ADS)

van den Heuvel, Quint; Matveev, Sergei; Drury, Martyn; Gress, Michael; Chinn, Ingrid; Davies, Gareth

2017-04-01

Diamond inclusions are potentially fundamental to understanding the formation conditions of diamond and the volatile cycles in the deep mantle. In order to fully understand the implications of the compositional information recorded by inclusions it is vital to know whether the inclusions are proto-, syn-, or epigenetic and the extent to which they have equilibrated with diamond forming fluids. In previous studies, the widespread assumption was made that the majority of diamond inclusions are syngenetic, based upon observation of cubo-octahedral morphology imposed on the inclusions. Recent work has reported the crystallographic relationship between inclusions and the host diamond to be highly complex and the lack of crystallographic relationships between inclusions and diamonds has led some to question the significance of imposed cubo-octahedral morphology. This study presents an integrated EBSD and CL study of 9 diamonds containing 20 pyropes, 2 diopsides, 1 forsterite and 1 rutile from the Jwaneng and Letlhakane kimberlite clusters, Botswana. A new method was developed to analyze the crystallographic orientation of the host diamond and the inclusions with EBSD. Diamonds plates were sequentially polished to expose inclusions at different levels in the diamond. CL imaging at different depths was performed in order to produce a 3D view of diamond growth zones around the inclusions. Standard diamond polishing techniques proved too aggressive for silicate inclusions as they were damaged to such a degree that EBSD measurements on the inclusions were impossible. The inclusions were milled with a Ga+ focused ion beam (FIB) at a 12° angle to clean the surface for EBSD measurements. Of the 24 inclusions, 9 have an imposed cubo-octahedral morphology. Of these inclusions, 6 have faces orientated parallel to diamond growth zones and/or appear to have nucleated on a diamond growth surface, implying syngenesis. In contrast, other diamonds record resorption events such that

Social Inclusion and Metrolingual Practices

ERIC Educational Resources Information Center

Otsuji, Emi; Pennycook, Alastair

2011-01-01

In this paper, we explore the implications of metrolingual language practices for how we understand social inclusion. A vision of social inclusion that includes bi- and multilingual capacities may comprise an appreciation of a diversity of languages other than English, and the skills and capabilities of multilingual language users, yet it is all…

Promoting Inclusive Education in Ghana

ERIC Educational Resources Information Center

Djietror, Beauty B. K.; Okai, Edward; Kwapong, Olivia A. T. Frimpong

2011-01-01

Inclusive education is critical for nation building. The government of Ghana has put in measures for promoting inclusion from basic through to tertiary level of education. Some of these measures include expansion of school facilities, implementation of the Free Compulsory Universal Basic Education (FCUBE); the change of policy on girls who drop…

Principals Influence Culture of Inclusion.

ERIC Educational Resources Information Center

King, Sophia

2000-01-01

Three New American High Schools (in Pittsburgh, Baltimore, and Greenbelt, Maryland) have successfully replaced the special-education culture of separation with a culture of inclusion. A large part of moving from self-contained practices to inclusion was helping students become self-advocates, so that the world outside school and home would not…

Deconstructing Normalisation: Clearing the Way for Inclusion.

ERIC Educational Resources Information Center

Culham, Andrew; Nind, Melanie

2003-01-01

This paper considers two major movements affecting the lives of people with intellectual disabilities: normalization and inclusion. It reviews the aims, processes, and outcomes of the normalization and social role valorization movement and explores its compatibility with inclusion. Lessons from normalization are applied to the inclusion movement.…

Teachers' Experience with Inclusive Education in Singapore

ERIC Educational Resources Information Center

Yeo, Lay See; Chong, Wan Har; Neihart, Maureen F.; Huan, Vivien S.

2016-01-01

Teachers' positive attitude is most critically and consistently associated with successful inclusion. However, little is known about teachers' first-hand encounters with inclusive education in Singapore. We present findings from a qualitative study on inclusion based on focus group interviews with 202 teachers from 41 resourced primary schools.…

Fluid Inclusions in Carbonaceous Chondrites

NASA Technical Reports Server (NTRS)

Saylor, J.; Zolensky, M. E.; Bodnar, R. J.; Le L.; Schwandt, C.

2001-01-01

Fluid inclusions are present in carbonaceous chondrites. Of the chondrites studied (CI1, CM1 and 2, CV3) fluid inclusions were found only in CM2s and CI1s, and by extrapolation are most likely to be found there in the future. Additional information is contained in the original extended abstract.

Inclusive Classes in Physical Education: Teachers' Difficulties

ERIC Educational Resources Information Center

Toloi, Gabriela Gallucci; Manzini, Eduardo José; Spoldaro, Diego Machado; Zacarias, Lucas Ventura

2016-01-01

The successful inclusion of students with special needs in physical education classes requires much planning and preparation. Lack of preparation of physical education teachers working in inclusive settings in Brazil has demonstrated the need for specialized training in strategies for implementing inclusion. The goal of this study was to identify,…

Inclusive Education: Examining Equity on Five Continents

ERIC Educational Resources Information Center

Artiles, Alfredo J., Ed.; Kozleski, Elizabeth B., Ed.; Waitoller, Federico R., Ed.

2011-01-01

Despite the impressive growth of inclusive education around the world, questions and considerations about equity have been neglected. This edited volume makes a major contribution to the field of inclusive education by analyzing equity concerns that have emerged from the implementation of inclusive education models in nine nations on five…

Class Inclusion and Role-Taking: Structural Mediation.

ERIC Educational Resources Information Center

Gash, Hugh

The mediational linkage between class inclusion and role-taking skills was investigated by studying the effects of a successive perspective-taking training technique on the consolidation of class inclusion structures. Sixty preoperational Irish boys were given two pretest measures of class inclusion and two of role-taking. They were then grouped…

Renal pathophysiologic role of cortical tubular inclusion bodies.

PubMed

Radi, Zaher A; Stewart, Zachary S; Grzemski, Felicity A; Bobrowski, Walter F

2013-01-01

Renal tubular inclusion bodies are rarely associated with drug administration. The authors describe the finding of renal cortical tubular intranuclear and intracytoplasmic inclusion bodies associated with the oral administration of a norepinephrine/serotonin reuptake inhibitor (NSRI) test article in Sprague-Dawley (SD) rats. Rats were given an NSRI daily for 4 weeks, and kidney histopathologic, ultrastructural pathology, and immunohistochemical examinations were performed. Round eosinophilic intranuclear inclusion bodies were observed histologically in the tubular epithelial cells of the renal cortex in male and female SD rats given the NSRI compound. No evidence of degeneration or necrosis was noted in the inclusion-containing renal cells. By ultrastructural pathology, inclusion bodies consisted of finely granular, amorphous, and uniformly stained nonmembrane-bound material. By immunohistochemistry, inclusion bodies stained positive for d-amino acid oxidase (DAO) protein. In addition, similar inclusion bodies were noted in the cytoplasmic tubular epithelial compartment by ultrastructural and immunohistochemical examination.  This is the first description of these renal inclusion bodies after an NSRI test article administration in SD rats. Such drug-induced renal inclusion bodies are rat-specific, do not represent an expression of nephrotoxicity, represent altered metabolism of d-amino acids, and are not relevant to human safety risk assessment.

Inclusive Education in Schools in Rural Areas

ERIC Educational Resources Information Center

Callado Moreno, J. Antonio; Molina Jaén, Ma. Dolores; Pérez Navío, Eufrasio; Rodríguez Moreno, Javier

2016-01-01

Since Spain decided to embark on the development of inclusive schooling, studies have taken place to see if the inclusive principle is being developed satisfactorily. Inclusive schooling implies that all students, regardless of their particular characteristics, may be taught in ordinary schools, and in the majority of cases receive help in the…

Pre-Service Educators' Attitudes towards Inclusive Education

ERIC Educational Resources Information Center

Mdikana, Andile; Ntshangase, Sibusiso; Mayekiso, Tokozile

2007-01-01

The inclusion of learners with special educational needs in general education is becoming more prevalent. As a result various special education researchers have begun to examine the success of inclusion, as well as the attitudes and beliefs of general educators towards the inclusion of learners with disabilities in the general education classroom.…

Creative Educational Practices for Inclusion

ERIC Educational Resources Information Center

Piske, Fernanda Hellen Ribeiro; Stoltz, Tania; Machado, Jarci

2014-01-01

Inclusion of gifted students depends on several aspects to happen in the school context, and one of the most important aspects to include these children at school is creative educational practices. Teaching with art is a good possibility to make children feel motivated to attend school. In the school context, the inclusion of these children could…

Do Melt Inclusions Answer Big Questions?

NASA Astrophysics Data System (ADS)

Hofmann, A. W.; Sobolev, A. V.

2009-12-01

In a pioneering paper, Sobolev and Shimizu (1993) demonstrated the existence of ultra-depleted melt inclusions in olivine phenocrysts in MORB. They interpreted these as evidence for the preservation of parental melts formed by progressive near-fractional melting. Subsequently many cases have been described where melt inclusions from single basalt samples display enormous chemical and isotopic heterogeneity. The interpretation of these observations hinges critically on whether such melt inclusions can faithfully preserve primary or parental melt composition. If they do, melt inclusion data can truly answer big questions from small-scale observations. If they do not, they answer rather small questions. Favoring the second possibility, Danyushevsky et al. (2004) have suggested that much of the observed variability of highly incompatible trace elements in melt inclusions “may not represent geologically significant melts, but instead reflect localized, grain-scale reaction processes within the magmatic plumbing system.” We disagree and show that this mechanism cannot, for example, explain isotopic heterogeneity measured in several suites of melt inclusions, nor does it not account for the presence of ultra-depleted melts and "ghost" plagioclase signatures in other inclusions. More recently, Spandler et al. (2007) have suggested on the basis of experimental evidence that diffusion rates for REE in olivine are so rapid that parental melt compositions in melt inclusions are rapidly falsified by diffusional exchange with (evolved) host lava. We show that the very fact that extreme chemical and isotopic heterogeneities are routinely preserved in melt inclusions demonstrates that this conclusion is unwarranted, either because residence times of the olivine phenocrysts are much shorter than assumed by Spandler et al. or because the high experimental diffusion rates are caused by an unknown experimental artifact. Although there is no obvious flaw in design and execution of

Is inclusive education a human right?

PubMed

Gordon, John-Stewart

2013-01-01

In this article, I question the general idea that inclusive education--i.e., to teach all students in one class--is a moral human right. The following discussion shows that the widespread view in disability studies that there is a moral human right to inclusive education can be reasonably called into question by virtue of the proposed counter arguments, but without denying that inclusive education is of utmost importance. Practically speaking, the legal human right to inclusive education is of great practical value for impaired students, and for their basic right to be free from discrimination in education, since their concern thereby gains great legal and moral force. But, theoretically speaking, this particular human right lacks an attainable consensus concerning proper moral justification. © 2013 American Society of Law, Medicine & Ethics, Inc.

Insights into Inclusive Education through a Small Finnish Case Study of an Inclusive School Context

ERIC Educational Resources Information Center

Tarr, Jane Mary; Tsokova, Diana; Takkunen, Ulla-Maija

2012-01-01

This study seeks to present data and discussion arising from a case study of a school in Finland renowned for its practice in the inclusion of learners with additional support requirements due to cognitive and physical disabilities. It aims to establish how the school staff understand their practice with inclusion through day-to-day professional…

Inclusive Schooling; Global Ideals and National Realities

ERIC Educational Resources Information Center

Winzer, Maggie; Mazurek, Kas

2009-01-01

In the past two decades, inclusive schooling for students with exceptional conditions has emerged in a range of national contexts to the extent that inclusion is now a world-wide movement. Major prompts arise from mandates and directives from international bodies such as UNESCO. However, the provision of education built on social inclusion places…

26 CFR 26.2642-1 - Inclusion ratio.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 14 2012-04-01 2012-04-01 false Inclusion ratio. 26.2642-1 Section 26.2642-1... GENERATION-SKIPPING TRANSFER TAX REGULATIONS UNDER THE TAX REFORM ACT OF 1986 § 26.2642-1 Inclusion ratio. (a) In general. Except as otherwise provided in this section, the inclusion ratio is determined by...

26 CFR 26.2642-1 - Inclusion ratio.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 14 2013-04-01 2013-04-01 false Inclusion ratio. 26.2642-1 Section 26.2642-1... GENERATION-SKIPPING TRANSFER TAX REGULATIONS UNDER THE TAX REFORM ACT OF 1986 § 26.2642-1 Inclusion ratio. (a) In general. Except as otherwise provided in this section, the inclusion ratio is determined by...

26 CFR 26.2642-1 - Inclusion ratio.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 14 2014-04-01 2013-04-01 true Inclusion ratio. 26.2642-1 Section 26.2642-1... GENERATION-SKIPPING TRANSFER TAX REGULATIONS UNDER THE TAX REFORM ACT OF 1986 § 26.2642-1 Inclusion ratio. (a) In general. Except as otherwise provided in this section, the inclusion ratio is determined by...

26 CFR 26.2642-1 - Inclusion ratio.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 14 2011-04-01 2010-04-01 true Inclusion ratio. 26.2642-1 Section 26.2642-1... GENERATION-SKIPPING TRANSFER TAX REGULATIONS UNDER THE TAX REFORM ACT OF 1986 § 26.2642-1 Inclusion ratio. (a) In general. Except as otherwise provided in this section, the inclusion ratio is determined by...

Measuring Inclusive Education Outcomes in Alberta, Canada

ERIC Educational Resources Information Center

Loreman, Tim

2014-01-01

This study details the results of a review of the academic and public sector literature on measuring inclusive education in large systems. It highlights some outcomes drawn from the international literature on inclusion that might be indicative of the presence and quality of inclusive education in an effort to develop a set of outcomes for…

Inclusive practices for children and youths with communication disorders. Ad Hoc Committee on Inclusion for students with Communication Disorders.

PubMed

1996-01-01

An array of inclusive service delivery models is recommended for the implementation of services to children and youths with communication disorders. Inclusive practices are intervention services that are based on the unique and specific needs of the individual, and provided in a context that is least restrictive. There are a variety of models through which inclusive practices can be provided, including a direct (pull-out) program, in classroom-based service delivery, community-based models, and consultative interventions. These models should be seen as flexible options that may change depending on student needs. The speech-language pathologist, in collaboration with parents, the student, teachers, support personnel, and administrators, is in the ideal position to decide the model or combination of models that best serves each individual student's communication needs. Implementation of inclusive practices requires consideration of multiple issues, including general education reform, cost effectiveness, and program efficacy. In addition, administrative and school system support, personnel qualifications, staff development, flexible scheduling, and the effects of inclusive practices on all learners need to be considered. At present, available research suggests guarded optimism for the effectiveness of inclusive practices. However, many critical questions have not yet been addressed and additional research is needed to assess the full impact of inclusive practices for students with communication disorders.

Co-producing social inclusion: the structure/agency conundrum.

PubMed

Clifton, A; Repper, J; Banks, D; Remnant, J

2013-08-01

There is a raft of policy guidelines indicating that mental health nurses should be increasing the social inclusion of mental health service users. Despite this there is no universally accepted definition of social inclusion and there is a dearth of empirical evidence on the successful outcome of increasing inclusion for mental health service users. Recognizing the lack of clarity surrounding the concept we have a produced a social inclusion framework to assist mental health professionals and service users to co-produce social inclusive outcomes. Although we agree that social inclusion can be a positive aspect of recovery, we question the extent to which mental health nurses and service users in co-production can overcome the social, economic and political structures that have created the social exclusion in the first place. An understanding and appreciation of the structure/agency conundrum is required if mental health nurses are to engage with service users in an attempt to co-produce socially inclusive outcomes. © 2012 John Wiley & Sons Ltd.

Elements of Inclusion: Findings from the Field

ERIC Educational Resources Information Center

McMaster, Christopher

2014-01-01

The Ministry of Education has set the target of 100% of New Zealand schools to be "mostly" inclusive by 2014. But what are the essential elements of inclusion? This paper explores essential core elements that allow inclusion to flourish. Based on an extensive time in the field as part of a year-long doctoral research project, these…

Genetics Home Reference: microvillus inclusion disease

MedlinePlus

... Citation on PubMed Khubchandani SR, Vohra P, Chitale AR, Sidana P. Microvillous inclusion disease--an ultrastructural diagnosis: ... O, Utermann G, Ruemmele FM, Huber LA, Janecke AR. MYO5B mutations cause microvillus inclusion disease and disrupt ...

Deformability of Oxide Inclusions in Tire Cord Steels

NASA Astrophysics Data System (ADS)

Zhang, Lifeng; Guo, Changbo; Yang, Wen; Ren, Ying; Ling, Haitao

2018-04-01

The deformation of oxide inclusions in tire cord steels during hot rolling was analyzed, and the factors influencing their deformability at high and low temperatures were evaluated and discussed. The aspect ratio of oxide inclusions decreased with the increasing reduction ratio of the steel during hot rolling owing to the fracture of the inclusions. The aspect ratio obtained after the first hot-rolling process was used to characterize the high-temperature deformability of the inclusions. The deformation first increased and then decreased with the increasing (MgO + Al2O3)/(SiO2 + MnO) ratio of the inclusions. It also increased with the decreasing melting temperatures of the inclusions. Young's modulus was used to evaluate the low-temperature deformability of the inclusions. An empirical formula was fitted to calculate the Young's moduli of the oxides using the mean atomic volume. The moduli values of the inclusions causing wire fracture were significantly greater than the average. To reduce fracture in tire cord steel wires during cold drawing, it is proposed that inclusions be controlled to those with high SiO2 content and extremely low Al2O3 content. This proposal is based on the hypothesis that the deformabilities of oxides during cold drawing are inversely proportional to their Young's moduli. The future study thus proposed includes an experimental confirmation for the abovementioned predictions.

Eshelby's problem of non-elliptical inclusions

NASA Astrophysics Data System (ADS)

Zou, Wennan; He, Qichang; Huang, Mojia; Zheng, Quanshui

2010-03-01

The Eshelby problem consists in determining the strain field of an infinite linearly elastic homogeneous medium due to a uniform eigenstrain prescribed over a subdomain, called inclusion, of the medium. The salient feature of Eshelby's solution for an ellipsoidal inclusion is that the strain tensor field inside the latter is uniform. This uniformity has the important consequence that the solution to the fundamental problem of determination of the strain field in an infinite linearly elastic homogeneous medium containing an embedded ellipsoidal inhomogeneity and subjected to remote uniform loading can be readily deduced from Eshelby's solution for an ellipsoidal inclusion upon imposing appropriate uniform eigenstrains. Based on this result, most of the existing micromechanics schemes dedicated to estimating the effective properties of inhomogeneous materials have been nevertheless applied to a number of materials of practical interest where inhomogeneities are in reality non-ellipsoidal. Aiming to examine the validity of the ellipsoidal approximation of inhomogeneities underlying various micromechanics schemes, we first derive a new boundary integral expression for calculating Eshelby's tensor field (ETF) in the context of two-dimensional isotropic elasticity. The simple and compact structure of the new boundary integral expression leads us to obtain the explicit expressions of ETF and its average for a wide variety of non-elliptical inclusions including arbitrary polygonal ones and those characterized by the finite Laurent series. In light of these new analytical results, we show that: (i) the elliptical approximation to the average of ETF is valid for a convex non-elliptical inclusion but becomes inacceptable for a non-convex non-elliptical inclusion; (ii) in general, the Eshelby tensor field inside a non-elliptical inclusion is quite non-uniform and cannot be replaced by its average; (iii) the substitution of the generalized Eshelby tensor involved in various

Inclusion Bodies Are a Site of Ebolavirus Replication

PubMed Central

Hoenen, Thomas; Shabman, Reed S.; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F.; Becker, Stephan

2012-01-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place. PMID:22915810

Inclusion bodies are a site of ebolavirus replication.

PubMed

Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

2012-11-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

A new barometer from stress fields around inclusions

NASA Astrophysics Data System (ADS)

Avadanii, Diana; Hansen, Lars; Wallis, David; Waters, David

2017-04-01

A key step in understanding geological and geodynamic processes is modelling the pressure-temperature paths of metamorphic rocks. Traditional thermobarometry relies on mineral assemblage equilibria and thermodynamic modelling to infer the pressures and temperatures of chemical equilibration. This approach requires the presence of specific mineral assemblages and compositions, which narrows its applicability. In this study we aim to develop a geobarometer based on mechanical interactions between inclusions and their host grains. Exhumation of minerals with inclusions causes heterogeneous residual stress fields due to the different, and often anisotropic, elastic properties of the inclusion and host. Recent studies measure residual mean stresses within inclusions using Raman spectroscopy and use those stresses as a barometer. In contrast, we map each component of the stress tensor around inclusions using high angular-resolution electron backscatter diffraction (HR-EBSD). This technique provides both higher spatial resolution and increased sensitivity to elastic strains relative to Raman spectroscopy. We focus on quartz inclusions in garnet, a common feature in metamorphic rocks. This assemblage also provides an opportunity to test our results with compositional thermobarometry. We analyse samples metamorphosed at pressures ranging from ˜ 300 MPa to ˜ 1600 MPa, as recorded by independent geobarometers. HR-EBSD reveals symmetric and lobate signals around inclusions, with elastic strains and residual stresses of the order 10-3 and ±102 -103 MPa, respectively. We solve Eshelby's problem for the 'inhomogeneous inclusion' case to simulate the elastic strain/stress field around an anisotropic ellipsoidal inclusion surrounded by an isotropic, homogeneous, infinite matrix. This model calculates the stress disturbances caused by differential expansion of an inclusion and host subjected to decompression. We additionally account for differential expansion related to cooling

Diagenetic palaeotemperatures from aqueous fluid inclusions: re- equilibration of inclusions in carbonate cements by burial heating.

USGS Publications Warehouse

Burruss, R.C.

1987-01-01

Calculations based on the observed behaviour of inclusions in fluorite under external confining P allows prediction of the T and depths of burial necessary to initiate re-equilibration of aqueous inclusions in the common size range 40-4 mu m. Heating of 20-60oC over the initial trapping T may cause errors of 10-20oC in the homogenization T. This suggests that re-equilibration may cause aqueous inclusions in carbonates to yield a poor record of their low-T history, but a useful record of the maximum T experienced by the host rock. Previous work suggests that inclusions containing petroleum fluids will be less susceptible to re-equilibration.This and the following six abstracts represent papers presented at a joint meeting of the Applied Mineralogy Group of the Mineralogical Society and the Petroleum Group of the Geological Society held in Newcastle upon Tyne in April 1986.-R.A.H.

Microcrystalline dolomite within massive Japan Sea methane hydrate: origin and development ascertained by inclusions within inclusions.

NASA Astrophysics Data System (ADS)

Snyder, G. T.; Kakizaki, Y.; Matsumoto, R.; Suzuki, Y.; Takahata, N.; Sano, Y.; Tanaka, K.; Tomaru, H.; Imajo, T.; Iguchi, A.

2017-12-01

Microcrystalline dolomite grains were recently discovered as inclusions within relatively pure massive gas hydrate recovered from the Joetsu Basin area of the Japan Sea. These grains presumably formed as a consequence of the highly saline conditions in fluid inclusions which developed between coalescing grain boundaries within the growing hydrate. Stable carbon and oxygen isotopic composition of the dolomite is consistent with crystal growth occurring within such fluids. In addition to stable isotopes, we investigate trends in Mg/Ca ratios of the grains as well as the composition of inclusions which exist within the dolomites. Preliminary research shows that these inclusions retain valuable information as to the conditions which existed at the time of formation, as well as the dynamics of these extensive hydrate deposits over time. This study was conducted under the commission from AIST as a part of the methane hydrate research project funded by METI (the Ministry of Economy, Trade and Industry, Japan).

Advancing diversity through inclusive excellence in nursing education.

PubMed

Bleich, Michael R; MacWilliams, Brent R; Schmidt, Bonnie J

2015-01-01

Nurse leaders call for a more diverse nursing workforce, but too few address the concept of inclusion as a recruitment and retention strategy or as part of improving the academic learning milieu. This article addresses organizational considerations of diversity and inclusion as part of the agenda established by the Association of American Colleges and Universities for inclusive excellence, building on the idea that academic environments only become excellent when an inclusive climate is reached. Six organizational strategies to inclusion are presented from the authors' experiences, some structural and others behavioral: admissions processes, invisibility, absence of community, promotion and tenure, exclusion, and tokenism. A call for structural and behavioral adaptions within nursing education to advance an inclusive excellence agenda is presented. Copyright © 2015 Elsevier Inc. All rights reserved.

Diversifying Geoscience by Preparing Faculty as Workshop Leaders to Promote Inclusive Teaching and Inclusive Geoscience Departments

NASA Astrophysics Data System (ADS)

Macdonald, H.; Manduca, C. A.; Beane, R. J.; Doser, D. I.; Ebanks, S. C.; Hodder, J.; McDaris, J. R.; Ormand, C. J.

2017-12-01

Efforts to broaden participation in the geosciences require that faculty implement inclusive practices in their teaching and their departments. Two national projects are building the capacity for faculty and departments to implement inclusive practices. The NAGT/InTeGrate Traveling Workshops Program (TWP) and the Supporting and Advancing Geoscience Education in Two-Year Colleges (SAGE 2YC) project each prepares a cadre of geoscience educators to lead workshops that provide opportunities for faculty and departments across the country to enhance their abilities to implement inclusive teaching practices and develop inclusive environments with the goal of increasing diversity in the geosciences. Both projects prepare faculty to design and lead interactive workshops that build on the research base, emphasize practical applications and strategies, enable participants to share their knowledge and experience, and include time for reflection and action planning. The curriculum common to both projects includes a framework of support for the whole student, supporting all students, data on diversity in the geosciences, and evidence-based strategies for inclusive teaching and developing inclusive environments that faculty and departments can implement. Other workshop topics include classroom strategies for engaging all students, addressing implicit bias and stereotype threat, and attracting diverse students to departments or programs and helping them thrive. Online resources for each project provide support beyond the workshops. The TWP brings together educators from different institutional types and experiences to develop materials and design a workshop offered to departments and organizations nationwide that request the workshop; the workshop leaders then customize the workshop for that audience. In SAGE 2YC, a team of leaders used relevant literature to develop workshop materials intended for re-use, and designed a workshop session for SAGE 2YC Faculty Change Agents, who

Arterial Stiffness Measured with the Cuff Oscillometric Method Is Predictive of Exercise Capacity in Patients with Cardiac Diseases.

PubMed

Tazawa, Yasushi; Mori, Nobuyoshi; Ogawa, Yoshiko; Ito, Osamu; Kohzuki, Masahiro

2016-06-01

Arterial stiffness is widely used in assessing arteriosclerosis in the background of increased cardiovascular events. Arteriosclerosis also causes reduction in exercise capacity, which is a most important prognostic factor in patients with cardiovascular disease; however, data on the association between arterial stiffness and exercise capacity are limited. Therefore, a simple and noninvasive measurement of arterial stiffness that reflects the central circulation and exercise capacity is needed. The arterial velocity pulse index (AVI) is a parameter of arterial stiffness measurable with the cuff oscillometric method; however, the clinical utility of this method is unclear. We aimed to evaluate the trend of AVI in patients with coronary artery disease (CAD), and the association between AVI and exercise capacity. A cross-sectional study of 116 patients with cardiac disease (34 CAD and 82 non-CAD patients) was performed. Non-CAD patients were those with any cardiac diseases who did not have proven CAD. The results showed that the AVI was significantly higher in CAD patients than non-CAD patients (P < 0.05, analysis of covariance). The AVI was inversely correlated with peakVO2 (r = -0.239, P < 0.05) and was a significant explanatory variable for peakVO2 in stepwise regression analysis (β = -14.62, t = -2.5, P < 0.05). These results indicate that the AVI is strongly associated with CAD and predictive of the exercise capacity in patients with cardiac diseases. We, therefore, propose that the cuff oscillometric method is clinically useful in evaluating arterial stiffness in patients with cardiac diseases, especially CAD.

Early-life lead exposure results in dose- and sex-specific effects on weight and epigenetic gene regulation in weanling mice

PubMed Central

Faulk, Christopher; Barks, Amanda; Liu, Kevin; Goodrich, Jaclyn M; Dolinoy, Dana C

2013-01-01

Aims Epidemiological and animal data suggest that the development of adult chronic conditions is influenced by early-life exposure-induced changes to the epigenome. This study investigates the effects of perinatal lead (Pb) exposure on DNA methylation and bodyweight in weanling mice. Materials & methods Viable yellow agouti (Avy) mouse dams were exposed to 0, 2.1, 16 and 32 ppm Pb acetate before conception through weaning. Epigenetic effects were evaluated by scoring coat color of Avy/a offspring and quantitative bisulfite sequencing of two retrotransposon-driven (Avy and CDK5 activator-binding protein intracisternal A particle element) and two imprinted (Igf2 and Igf2r) loci in tail DNA. Results Maternal blood Pb levels were below the limit of detection in controls, and 4.1, 25.1 and 32.1 μg/dl for each dose, respectively. Pb exposure was associated with a trend of increased wean bodyweight in males (p = 0.03) and altered coat color in Avy/a offspring. DNA methylation at Avy and the CDK5 activator-binding protein intracisternal A-particle element was significantly different from controls following a cubic trend (p = 0.04; p = 0.01), with male-specific effects at the Avy locus. Imprinted genes did not shift in methylation across exposures. Conclusion Dose- and sex-specific responses in bodyweight and DNA methylation indicate that Pb acts on the epigenome in a locus-specific fashion, dependent on the genomic feature hosting the CpG site of interest, and that sex is a factor in epigenetic response. PMID:24059796

Effective interactions between inclusions in an active bath

NASA Astrophysics Data System (ADS)

Zaeifi Yamchi, Mahdi; Naji, Ali

2017-11-01

We study effective two- and three-body interactions between non-active colloidal inclusions in an active bath of chiral or non-chiral particles, using Brownian dynamics simulations within a standard, two-dimensional model of disk-shaped inclusions and active particles. In a non-chiral active bath, we first corroborate previous findings on effective two-body repulsion mediated between the inclusions by elucidating the detailed non-monotonic features of the two-body force profiles, including a primary maximum and a secondary hump at larger separations that was not previously reported. We then show that these features arise directly from the formation, and sequential overlaps, of circular layers (or "rings") of active particles around the inclusions, as the latter are brought to small surface separations. These rings extend to radial distances of a few active-particle radii from the surface of inclusions, giving the hard-core inclusions relatively thick, soft, repulsive "shoulders," whose multiple overlaps then enable significant (non-pairwise) three-body forces in both non-chiral and chiral active baths. The resulting three-body forces can even exceed the two-body forces in magnitude and display distinct repulsive and attractive regimes at intermediate to large self-propulsion strengths. In a chiral active bath, we show that, while active particles still tend to accumulate at the immediate vicinity of the inclusions, they exhibit strong depletion from the intervening region between the inclusions and partial depletion from relatively thick, circular zones further away from the inclusions. In this case, the effective, predominantly repulsive interactions between the inclusions turn to active, chirality-induced, depletion-type attractions, acting over an extended range of separations.

Inclusion bodies of aggregated hemosiderins in liver macrophages.

PubMed

Hayashi, Hisao; Tatsumi, Yasuaki; Wakusawa, Shinya; Shigemasa, Ryota; Koide, Ryoji; Tsuchida, Ken-Ichi; Morotomi, Natsuko; Yamashita, Tetsuji; Kumagai, Kotaro; Ono, Yukiya; Hayashi, Kazuhiko; Ishigami, Masatoshi; Goto, Hidemi; Kato, Ayako; Kato, Koichi

2017-12-01

Hemosiderin formation is a structural indication of iron overload. We investigated further adaptations of the liver to excess iron. Five patients with livers showing iron-rich inclusions larger than 2 µm were selected from our database. The clinical features of patients and structures of the inclusions were compared with those of 2 controls with mild iron overload. All patients had severe iron overload with more than 5000 ng/mL of serum ferritin. Etiologies were variable, from hemochromatosis to iatrogenic iron overload. Their histological stages were either portal fibrosis or cirrhosis. Inclusion bodies were ultra-structurally visualized as aggregated hemosiderins in the periportal macrophages. X-ray analysis always identified, in addition to a large amount of iron complexes including oxygen and phosphorus, a small amount of copper and sulfur in the mosaic matrixes of inclusions. There were no inclusions in the control livers. Inclusion bodies, when the liver is loaded with excess iron, may appear in the macrophages as isolated organella of aggregated hemosiderins. Trace amounts of copper-sulfur complexes were always identified in the mosaic matrices of the inclusions, suggesting cuproprotein induction against excess iron. In conclusion, inclusion formation in macrophages may be an adaptation of the liver loaded with excess iron.

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.

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane.

PubMed

Ukai, Hirofumi; Araki, Yasuhiro; Kira, Shintaro; Oikawa, Yu; May, Alexander I; Noda, Takeshi

2018-04-01

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane

PubMed Central

Ukai, Hirofumi; Araki, Yasuhiro; Kira, Shintaro; Oikawa, Yu; May, Alexander I.

2018-01-01

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1. PMID:29698392

Valuing Student Teachers' Perspectives: Researching Inclusively in Inclusive Education?

ERIC Educational Resources Information Center

Black-Hawkins, Kristine; Amrhein, Bettina

2014-01-01

This paper considers how engaging with the principles of inclusive research can enhance research studies that set out to understand the experiences of student teachers on initial teacher education programmes. It does so by describing the methodological development of an on-going study of student teachers' perspectives on working with diverse…

Inclusion "All Present and Correct?" A Critical Analysis of New Labour's Inclusive Education Policy in England

ERIC Educational Resources Information Center

Hodkinson, Alan

2013-01-01

Drawing on Derrida this paper considers how inclusive education in England was defined and operationalised within New Labour's educational policy and by those teachers who reconstructed this policy within the confines of schools and individual classrooms. The paper has two critical ambitions. First it argues that the epistemology of inclusion was…

Solutions for Dynamic Variants of Eshelby's Inclusion Problem

NASA Astrophysics Data System (ADS)

Michelitsch, Thomas M.; Askes, Harm; Wang, Jizeng; Levin, Valery M.

The dynamic variant of Eshelby's inclusion problem plays a crucial role in many areas of mechanics and theoretical physics. Because of its mathematical complexity, dynamic variants of the inclusion problems so far are only little touched. In this paper we derive solutions for dynamic variants of the Eshelby inclusion problem for arbitrary scalar source densities of the eigenstrain. We study a series of examples of Eshelby problems which are of interest for applications in materials sciences, such as for instance cubic and prismatic inclusions. The method which covers both the time and frequency domain is especially useful for dynamically transforming inclusions of any shape.

Implementation of a Post-Secondary Inclusive Recreation Program

ERIC Educational Resources Information Center

Brown, Chris; Johnson, John David; Elsawy, Ibrahim Z.

2017-01-01

As individuals with intellectual or developmental disabilities become increasingly involved in post-secondary educational opportunities, it becomes the responsibility of the institution to provide inclusive recreation opportunities. This manuscript incorporates Sugermans' (2001) Model of Inclusive Facilitation into an inclusive recreation program…

Exploring the Landscape of Inclusion: Profiles of Inclusive versus Segregated School Districts in the United States

ERIC Educational Resources Information Center

Marks, Susan Unok; Kurth, Jennifer A.; Bartz, Jody Marie

2014-01-01

Although inclusive education has been increasing in frequency for students with disabilities in the United States, for many students, the opportunity to be educated with their peers without disabilities continues to be out of reach despite decades of efforts by those promoting the vision of inclusion. This exploratory case study used interviews…

The Academic Achievement of Elementary Level General Education Students in Inclusion Classrooms versus Non-Inclusion Classrooms

ERIC Educational Resources Information Center

Surgener, Gena F.

2016-01-01

This research study was conducted to examine the effects of the academic achievement of elementary level general education students in the inclusion classroom setting versus the general education students in the non-inclusion classroom in a large suburban school district in Tennessee as measured by third, fourth, and fifth grade mathematics and…

Volatile loss during homogenization of lunar melt inclusions

NASA Astrophysics Data System (ADS)

Ni, Peng; Zhang, Youxue; Guan, Yunbin

2017-11-01

Volatile abundances in lunar mantle are critical factors to consider for constraining the model of Moon formation. Recently, the earlier understanding of a ;dry; Moon has shifted to a fairly ;wet; Moon due to the detection of measurable amount of H2O in lunar volcanic glass beads, mineral grains, and olivine-hosted melt inclusions. The ongoing debate on a ;dry; or ;wet; Moon requires further studies on lunar melt inclusions to obtain a broader understanding of volatile abundances in the lunar mantle. One important uncertainty for lunar melt inclusion studies, however, is whether the homogenization of melt inclusions would cause volatile loss. In this study, a series of homogenization experiments were conducted on olivine-hosted melt inclusions from the sample 74220 to evaluate the possible loss of volatiles during homogenization of lunar melt inclusions. Our results suggest that significant loss of H2O could occur even during minutes of homogenization, while F, Cl and S in the inclusions remain unaffected. We model the trend of H2O loss in homogenized melt inclusions by a diffusive hydrogen loss model. The model can reconcile the observed experimental data well, with a best-fit H diffusivity in accordance with diffusion data explained by the ;slow; mechanism for hydrogen diffusion in olivine. Surprisingly, no significant effect for the low oxygen fugacity on the Moon is observed on the diffusive loss of hydrogen during homogenization of lunar melt inclusions under reducing conditions. Our experimental and modeling results show that diffusive H loss is negligible for melt inclusions of >25 μm radius. As our results mitigate the concern of H2O loss during homogenization for crystalline lunar melt inclusions, we found that H2O/Ce ratios in melt inclusions from different lunar samples vary with degree of crystallization. Such a variation is more likely due to H2O loss on the lunar surface, while heterogeneity in their lunar mantle source is also a possibility. A

[Separation of gamma linolenic acid from evening primrose oil with urea inclusion--orthogonal experiment of optimizing technological parameters and observation of urea inclusion compound I].

PubMed

Wang, Hua; Ling, Man; Xue, Gang; Liu, Fengxia; Guo, Shuxian

2010-05-01

The influence on the urea inclusion compound under different conditions (allocated proportion, time of inclusion, temperature of inclusion) were studied through the orthogonal test, and theoretical reference of urea inclusion process for further optimization wound be offered. The orthogonal experiment was adopted, and microscope was used to observe the shape, aperture size of the urea inclusion compound under different technological parameters, the GC was employed to inspect the purity of GLA. The results indicated that the ratio of fatty acids and urea, inclusion of temperature, time of inclusion had great effect on urea inclusion compound. The three factors and its interactions significantly affected the purity of GLA. The results also showed that the best process was that the ratio of fatty acids and urea was 1 : 3, temperature of inclusion was--15 degrees C, time of inclusion was 24 h. Under the best condition, the purity of GLA reach up to 95.575 9%; and it is feasible to observe the shape and the amount of the urea inclusion compound to reflect and guide the urea inclusion technology.

Isolation of cell-free bacterial inclusion bodies.

PubMed

Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

2010-09-17

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9.

PubMed

Zhang, Jingbo; Baetz, Ulrike; Krügel, Undine; Martinoia, Enrico; De Angeli, Alexis

2013-10-01

Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.

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.

Intranuclear inclusions of meningioma associated with abnormal cytoskeletal protein expression.

PubMed

Yoshida, T; Hirato, J; Sasaki, A; Yokoo, H; Nakazato, Y; Kurachi, H

1999-01-01

We describe a case of meningothelial meningioma with a large number of intranuclear inclusions. Morphologically, these are divided into cytoplasmic inclusions and nuclear vacuoles. The cytoplasmic inclusion has a limiting membrane with cell organelles and filaments. Inclusions of this type are generally eosinophilic, like the cytoplasm. However, there are many inclusions that are more eosinophilic than the cytoplasm or that have a ground-glass appearance. Some of them may contain fine or coarse granules. On the other hand, the nuclear vacuole lacks a limiting membrane and appears empty. In most of the inclusions of this type, there is a faintly basophilic substance in the margin. Generally, the cytoplasmic inclusions are as immunopositive as cytoplasm with vimentin, but some of these cytoplasmic inclusions are more reactive. Under the electron microscope, abnormal aggregation of intermediate filaments is recognized in the cytoplasmic inclusions. It is considered that a strong reaction of cytoplasmic inclusions with vimentin immunostaining is due to abnormal aggregation of intermediate filaments. The present study distinctly demonstrates abnormal localization of intermediate filaments in the cytoplasmic inclusions, and it is suggested that the cytoskeleton participates in the evolution of the cytoplasmic inclusions.

Inclusion: Something More Than Sitting Together

ERIC Educational Resources Information Center

Bulbul, Mustafa Sahin

2011-01-01

Awareness about students with learning difficulty brought us to inclusive learning environments. The acceptance was to build collaborative atmospheres in the class. Unfortunately, when teachers are not enthusiastic and adequate to develop the interaction, the inclusive learning environment never occurs. This conclusion bases on this studies…

Irish Mathematics Teachers' Attitudes towards Inclusion

ERIC Educational Resources Information Center

Whitty, Elaine; Clarke, Marie

2012-01-01

This paper through the theoretical framework of constructive attitude theory explores mathematics teachers' attitudes and pedagogical strategies with reference to inclusive practice. The authors argue that though teachers may have formed positive inclusive attitudes, the translation of these into practice does not always occur and poses…

Culturally Inclusive Curriculum in Higher Education

ERIC Educational Resources Information Center

Pridham, Bruce; Martin, Dona; Walker, Kym; Rosengren, Rosie; Wadley, Danielle

2015-01-01

The Australian National Program Standards for Teacher Education prioritises knowledge of culturally inclusive practices and challenges the educational community to present research on well-structured, inclusive, cross-curricula education partnerships. This article meets this challenge as it explores a core unit of work for undergraduate teachers…

How Does Supervision Support Inclusive Teacherhood?

ERIC Educational Resources Information Center

Alila, Sanna; Määttä, Kaarina; Uusiautti, Satu

2016-01-01

Supervision is a multidimensional concept and phenomenon. In this study, the advantages of supervision and its development in inclusive teacherhood was studied. Inclusive teacherhood means a teacher's professional development and the school culture's change toward participatory school for all students. The study analyzed the views of supervisors…

Inclusive Education in Higher Education: Challenges and Opportunities

ERIC Educational Resources Information Center

Moriña, Anabel

2017-01-01

Implementing the principles of inclusive education within higher education can be challenging. Inclusive education was originally developed for younger students, prior to its application within higher education. However, as more students with disabilities successfully complete their early schooling, the need to move towards inclusive practices…

Agouti signaling protein stimulates cell division in "viable yellow" (A vy/a) mouse liver

USDA-ARS?s Scientific Manuscript database

Enhanced linear growth, hyperplasia, and tumorigenesis are well-known characteristics of "viable yellow" agouti Avy/- mice (1); however, the functional basis for this aspect of the phenotype is unknown. In the present study, we ascertained whether agouti signaling protein (ASIP) levels in Avy/a or a...

BD LSRFortessa for Tulane DoD ID and Cancer Projects

DTIC Science & Technology

2015-01-21

Influenza Antivirals" PI: Patrick Iversen, AVI BioPharma DTRA-TMTI (Subcontract) "Rapid Development of Dengue Antivirals" PI: Patrick Iversen, AVI... BioPharma (PI) Lisa Morici, PhD: Defense Threat Reduction Agency HDTRA 1-13-C-0002 "Outer membrane vesicle vaccine-mediated protection against

26 CFR 26.2642-5 - Finality of inclusion ratio.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 14 2014-04-01 2013-04-01 true Finality of inclusion ratio. 26.2642-5 Section...-5 Finality of inclusion ratio. (a) Direct skips. The inclusion ratio applicable to a direct skip...) Other GSTs. With respect to taxable distributions and taxable terminations, the inclusion ratio for a...

26 CFR 26.2642-5 - Finality of inclusion ratio.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 14 2012-04-01 2012-04-01 false Finality of inclusion ratio. 26.2642-5 Section...-5 Finality of inclusion ratio. (a) Direct skips. The inclusion ratio applicable to a direct skip...) Other GSTs. With respect to taxable distributions and taxable terminations, the inclusion ratio for a...

26 CFR 26.2642-5 - Finality of inclusion ratio.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 14 2013-04-01 2013-04-01 false Finality of inclusion ratio. 26.2642-5 Section...-5 Finality of inclusion ratio. (a) Direct skips. The inclusion ratio applicable to a direct skip...) Other GSTs. With respect to taxable distributions and taxable terminations, the inclusion ratio for a...

26 CFR 26.2642-5 - Finality of inclusion ratio.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 14 2011-04-01 2010-04-01 true Finality of inclusion ratio. 26.2642-5 Section...-5 Finality of inclusion ratio. (a) Direct skips. The inclusion ratio applicable to a direct skip...) Other GSTs. With respect to taxable distributions and taxable terminations, the inclusion ratio for a...

26 CFR 26.2642-5 - Finality of inclusion ratio.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 14 2010-04-01 2010-04-01 false Finality of inclusion ratio. 26.2642-5 Section...-5 Finality of inclusion ratio. (a) Direct skips. The inclusion ratio applicable to a direct skip...) Other GSTs. With respect to taxable distributions and taxable terminations, the inclusion ratio for a...

Language and Social Inclusion: Unexplored Aspects of Intercultural Communication

ERIC Educational Resources Information Center

Musgrave, Simon; Bradshaw, Julie

2014-01-01

Social inclusion policy in Australia has largely ignored key issues of communication for linguistic minorities, across communities and with the mainstream community. In the (now disbanded) Social Inclusion Board's reports (e.g., Social Inclusion Unit, 2009), the emphasis is on the economic aspects of inclusion, while little attention has been paid…

Effective transient behaviour of inclusions in diffusion problems

NASA Astrophysics Data System (ADS)

Brassart, Laurence; Stainier, Laurent

2018-06-01

This paper is concerned with the effective transport properties of heterogeneous media in which there is a high contrast between the phase diffusivities. In this case the transient response of the slow phase induces a memory effect at the macroscopic scale, which needs to be included in a macroscopic continuum description. This paper focuses on the slow phase, which we take as a dispersion of inclusions of arbitrary shape. We revisit the linear diffusion problem in such inclusions in order to identify the structure of the effective (average) inclusion response to a chemical load applied on the inclusion boundary. We identify a chemical creep function (similar to the creep function of viscoelasticity), from which we construct estimates with a reduced number of relaxation modes. The proposed estimates admit an equivalent representation based on a finite number of internal variables. These estimates allow us to predict the average inclusion response under arbitrary time-varying boundary conditions at very low computational cost. A heuristic generalisation to concentration-dependent diffusion coefficient is also presented. The proposed estimates for the effective transient response of an inclusion can serve as a building block for the formulation of multi-inclusion homogenisation schemes.

Radial scaling in inclusive jet production at hadron colliders

NASA Astrophysics Data System (ADS)

Taylor, Frank E.

2018-03-01

Inclusive jet production in p-p and p ¯ -p collisions shows many of the same kinematic systematics as observed in single-particle inclusive production at much lower energies. In an earlier study (1974) a phenomenology, called radial scaling, was developed for the single-particle inclusive cross sections that attempted to capture the essential underlying physics of pointlike parton scattering and the fragmentation of partons into hadrons suppressed by the kinematic boundary. The phenomenology was successful in emphasizing the underlying systematics of the inclusive particle productions. Here we demonstrate that inclusive jet production at the Large Hadron Collider (LHC) in high-energy p-p collisions and at the Tevatron in p ¯ -p inelastic scattering shows similar behavior. The ATLAS inclusive jet production plotted as a function of this scaling variable is studied for √s of 2.76, 7 and 13 TeV and is compared to p ¯ -p inclusive jet production at 1.96 TeV measured at the CDF and D0 at the Tevatron and p-Pb inclusive jet production at the LHC ATLAS at √sNN=5.02 TeV . Inclusive single-particle production at Fermi National Accelerator Laboratory fixed target and Intersecting Storage Rings energies are compared to inclusive J /ψ production at the LHC measured in ATLAS, CMS and LHCb. Striking common features of the data are discussed.

Design and control of six degree-of-freedom active vibration isolation table

NASA Astrophysics Data System (ADS)

Hong, Jinpyo; Park, Kyihwan

2010-03-01

A six-axis active vibration isolation system (AVIS) is designed by using the direct driven guide and ball contact mechanisms in order to have no cross-coupling between actuators. The point contact configuration gives an advantage of having an easy assembly of eight voice coil actuators to an upper and a base plate. A voice coil actuator is used since it can provide a large displacement and sufficient bandwidth required for vibration control. The AVIS is controlled considering the effect of flexible vibration mode in the upper plate and velocity sensor dynamics. A loop shaping technique and phase margin condition are applied to design a vibration controller. The performances of the AVIS are investigated in the frequency domain and finally validated by comparing with the passive isolation system. The scanning profiles of the specimen are compared together by using the atomic force microscope. The robustness of the AVIS is verified by showing the impulse response.

Design and control of six degree-of-freedom active vibration isolation table.

PubMed

Hong, Jinpyo; Park, Kyihwan

2010-03-01

A six-axis active vibration isolation system (AVIS) is designed by using the direct driven guide and ball contact mechanisms in order to have no cross-coupling between actuators. The point contact configuration gives an advantage of having an easy assembly of eight voice coil actuators to an upper and a base plate. A voice coil actuator is used since it can provide a large displacement and sufficient bandwidth required for vibration control. The AVIS is controlled considering the effect of flexible vibration mode in the upper plate and velocity sensor dynamics. A loop shaping technique and phase margin condition are applied to design a vibration controller. The performances of the AVIS are investigated in the frequency domain and finally validated by comparing with the passive isolation system. The scanning profiles of the specimen are compared together by using the atomic force microscope. The robustness of the AVIS is verified by showing the impulse response.

40 CFR 35.133 - Programs eligible for inclusion.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Programs eligible for inclusion. 35.133... Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible, in accordance with appropriation acts, for inclusion in a...

40 CFR 35.133 - Programs eligible for inclusion.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Programs eligible for inclusion. 35.133... Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible, in accordance with appropriation acts, for inclusion in a...

40 CFR 35.133 - Programs eligible for inclusion.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Programs eligible for inclusion. 35.133... Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible, in accordance with appropriation acts, for inclusion in a...

40 CFR 35.133 - Programs eligible for inclusion.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Programs eligible for inclusion. 35.133... Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible, in accordance with appropriation acts, for inclusion in a...

A Perspective of Inclusion: Challenges for the Future

ERIC Educational Resources Information Center

Braunsteiner, Maria-Luise; Mariano-Lapidus, Susan

2014-01-01

The term, "inclusion," particularly in the educational setting, is still based on a deficit view. Perceptions of "dis"-ability create barriers to true inclusion and are often reinforced through higher education training programs. To promote inclusive values, acceptance of individual and cultural differences must be included in…

40 CFR 35.133 - Programs eligible for inclusion.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Programs eligible for inclusion. 35.133... Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible, in accordance with appropriation acts, for inclusion in a...

Inclusive Education: Teacher Perspectives from Malaysia

ERIC Educational Resources Information Center

Bailey, Lucy; Nomanbhoy, Alefiya; Tubpun, Tida

2015-01-01

This paper reports on a survey of views of inclusive education expressed by nearly 300 Malaysian primary school teachers involved in remedial literacy and numeracy education under the country's Literacy and Numeracy Strategy. Overall, the views expressed were positive towards the principle of inclusion. However, despite common professional…

Students' Perspectives on LGBTQ-Inclusive Curriculum

ERIC Educational Resources Information Center

Snapp, Shannon D.; Burdge, Hilary; Licona, Adela C.; Moody, Raymond L.; Russell, Stephen T.

2015-01-01

Implementing curriculum that is inclusive of lesbian, gay, bisexual, transgender, queer, and questioning (LGBTQ) people has the potential to create an equitable learning environment. In order to learn more about students' experiences of LGBTQ-inclusive curriculum, 26 high school students with diverse racial/ethnic, sexual, and gender identities…

Moving Forward in Inclusive Education Research

ERIC Educational Resources Information Center

Erten, Ozlem; Savage, Robert Samuel

2012-01-01

This paper aims to address conceptual and methodological challenges of doing research in the field of inclusive education and revisit school effectiveness research literature to inform future research. First, we present the rationale for inclusive education and briefly review the evolution of special needs education. Then, we discuss limitations…

Developing Inclusive Practice in Scotland: The National Framework for Inclusion

ERIC Educational Resources Information Center

Barrett, Louise; Beaton, Mhairi; Head, George; McAuliffe, Lisa; Moscardini, Lio; Spratt, Jennifer; Sutherland, Margaret

2015-01-01

This paper reports on the collaborative development of a "National Framework for Inclusion" under the auspices of the Scottish Teacher Education Committee by a working party representing each of the Scottish Universities providing initial teacher education. Recent research, international legislation and Scottish education policy have…

Towards revealing the structure of bacterial inclusion bodies.

PubMed

Wang, Lei

2009-01-01

Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6-12 nm, they are comprised of residue-specific cross-beta structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

PubMed

Fabrizio, Paola; Hoon, Shawn; Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D

2010-07-15

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Interaction Among Inhomogeneities and Inclusions.

DTIC Science & Technology

1985-01-22

Some Recent Advances, ed. by X. Markenscoff, ASME, AMD, 63 (1984) 59-68. N. Kinoshita and T. Mura, " Eigenstrain Problems in a Finite Rody", SIAM J...terms of the free expansion strain ( eigenstrain ), the elastic moduli of the rock and the solidified demolition agent, and the fracture stress of rock...found an unexpected result for a sliding inclusion. It is found that when an ellipsoidal inclusion undergoes a uniform shear eigenstrain (non- elastic

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs)

PubMed Central

Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

2016-01-01

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs. PMID:27551866

Molecular and functional characterization of vacuolar-ATPase from the American dog tick Dermacentor variabilis.

PubMed

Petchampai, N; Sunyakumthorn, P; Guillotte, M L; Thepparit, C; Kearney, M T; Mulenga, A; Azad, A F; Macaluso, K R

2014-02-01

Vacuolar (V)-ATPase is a proton-translocating enzyme that acidifies cellular compartments for various functions such as receptor-mediated endocytosis, intracellular trafficking and protein degradation. Previous studies in Dermacentor variabilis chronically infected with Rickettsia montanensis have identified V-ATPase as one of the tick-derived molecules transcribed in response to rickettsial infection. To examine the role of the tick V-ATPase in tick-Rickettsia interactions, a full-length 2887-bp cDNA (2532-bp open reading frame) clone corresponding to the transcript of the V0 domain subunit a of D. variabilis V-ATPase (DvVATPaseV0a) gene encoding an 843 amino acid protein with an estimated molecular weight of ~96 kDa was isolated from D. variabilis. Amino acid sequence analysis of DvVATPaseV0a showed the highest similarity to VATPaseV0a from Ixodes scapularis. A potential N-glycosylation site and eight putative transmembrane segments were identified in the sequence. Western blot analysis of tick tissues probed with polyclonal antibody raised against recombinant DvVATPaseV0a revealed the expression of V-ATPase in the tick ovary. Transcriptional profiles of DvVATPaseV0a demonstrated a greater mRNA expression in the tick ovary, compared with the midgut and salivary glands; however, the mRNA level in each of these tick tissues remained unchanged after infection with R. montanensis for 1 h. V-ATPase inhibition bioassays resulted in a significant decrease in the ability of R. montanensis to invade tick cells in vitro, suggesting a role of V-ATPase in rickettsial infection of tick cells. Characterization of tick-derived molecules involved in rickettsial infection is essential for a thorough understanding of rickettsial transmission within tick populations and the ecology of tick-borne rickettsial diseases. © 2013 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

PubMed

Tang, Yujin; Wang, Ruipu; Gong, Peijie; Li, Shuxiu; Wang, Yuejin; Zhang, Chaohong

2016-01-01

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

Orientation relationship between diamond and magnesiochromite inclusions

NASA Astrophysics Data System (ADS)

Alvaro, Matteo; Angel, Ross; Nimis, Paolo; Milani, Sula; Harris, Jeff; Nestola, Fabrizio

2017-04-01

The correct determination of the relative crystallographic orientations of single crystals has many applications. When single crystals undergo phase transitions, especially at high pressures, the relative orientations of the two phases yields insights into transition mechanisms (Dobson et al 2013). On the other hand, determination of the crystallographic orientations of minerals included in diamonds can provide insights into the mechanisms of their entrapment and the timing of their formation relative to the host diamond (e.g. Nestola et al. 2014, Milani et al. 2016). The reported occurrence of non-trivial orientations for some minerals in diamonds, suggesting an epitaxial relationship, has long been considered to reflect contemporaneous growth of the diamond and the inclusion (e.g. syngenesis). Correct interpretation of such orientations requires (i) a statistically significant crystallographic data set for single and multiple inclusions in a large number of diamonds, and (ii) a robust data-processing method, capable of removing ambiguities derived from the high symmetry of the diamond and the inclusion. We have developed a software to perform such processing (OrientXplot, Angel et al. 2015), starting from crystallographic orientation matrixes obtained by X-ray diffractometry or EBSD data. Previous studies of inclusions in lithospheric diamonds, by single-crystal X-ray diffraction and EBSD, indicate a wide variety in the orientations of different inclusion phases with respect to their diamond host (Futergendler & Frank-Kamenetsky 1961; Frank-Kamenetsky 1964; Wiggers de Vries et al. 2011; Nestola et al. 2014, Milani et al. 2016). For example, olivine inclusions in lithospheric diamonds from Udachnaya do not show any preferred orientations with respect to their diamond hosts, but multiple inclusions in a single diamond often show very similar orientations within few degrees. In the present work on magnesiochromite inclusions in diamonds from Udachnaya, there is a

40 CFR 35.533 - Programs eligible for inclusion.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Programs eligible for inclusion. 35.533... § 35.533 Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible for inclusion in a Performance Partnership Grant are...

40 CFR 35.533 - Programs eligible for inclusion.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Programs eligible for inclusion. 35.533... § 35.533 Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible for inclusion in a Performance Partnership Grant are...

40 CFR 35.533 - Programs eligible for inclusion.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Programs eligible for inclusion. 35.533... § 35.533 Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible for inclusion in a Performance Partnership Grant are...

40 CFR 35.533 - Programs eligible for inclusion.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Programs eligible for inclusion. 35.533... § 35.533 Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible for inclusion in a Performance Partnership Grant are...

40 CFR 35.533 - Programs eligible for inclusion.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Programs eligible for inclusion. 35.533... § 35.533 Programs eligible for inclusion. (a) Eligible programs. Except as provided in paragraph (b) of this section, the environmental programs eligible for inclusion in a Performance Partnership Grant are...

Effect of inclusions on heterogeneous crack nucleation in nanocomposites

NASA Astrophysics Data System (ADS)

Gutkin, M. Yu.; Ovid'Ko, I. A.; Skiba, N. V.

2007-02-01

A two-dimensional theoretical model is proposed for the heterogeneous nucleation of a grain-boundary nanocrack in a nanocomposite consisting of a nanocrystalline matrix and nanoinclusions whose elastic moduli are identical to those of the matrix. The inclusions have the form of rods with a rectangular cross section and undergo dilatation eigenstrain induced by the differences in the lattice parameters and thermal expansion coefficients of the matrix and inclusions. In terms of the model, a mode-I-II nanocrack nucleates at the negative disclination of a biaxial dipole consisting of wedge grain-boundary (or junction) disclinations; then, the nanocrack opens along a grain boundary and reaches an inclusion boundary. Depending on the relative positions and orientations of the initial segment of the nanocrack and the inclusion, the nanocrack can either penetrate into the inclusion or bypass it along the matrix-inclusion interface. The nanocrack nucleation probability increases near an inclusion with negative (compressive) dilatation eigenstrain. A decrease in the inclusion size decreases (increases) the probability of a crack opening along the interface if the dilatation eigenstrain is negative (positive).

Effective Leadership Makes Schools Truly Inclusive

ERIC Educational Resources Information Center

McLeskey, James; Waldron, Nancy L.

2015-01-01

There's been much commitment and extensive legislation intended to make schools inclusive for all students but not much real progress in improving student outcomes. The authors review and assess several schools that have succeeded at making schools inclusive and effective for all students, including those with disabilities and draw some inferences…

Multiple Disabilities: Is Rural Inclusion Possible?

ERIC Educational Resources Information Center

Cates, Dennis L.; Smiley, Frederick M.

This paper focuses on the difficulties faced by rural school districts in their efforts to serve children with severe multiple disabilities. Both historic and contemporary views on mainstreaming and inclusion of students with multiple disabilities are presented. Concerns of educators about the inclusion of such students center around the amount of…

Early Childhood Inclusion: Focus on Change.

ERIC Educational Resources Information Center

Guralnick, Michael J., Ed.

Early childhood inclusion is a field characterized by a philosophy and practice that encourages full participation of children with disabilities and their families in everyday activities with their typically developing peers. Noting that success in inclusion endeavors requires substantial changes in the way our society thinks, feels, and acts,…

Fostering Social Acceptance in Inclusive Classrooms

ERIC Educational Resources Information Center

Wiener, Judith

2009-01-01

The mere presence of students with learning disabilities in general education classrooms is not inclusion. Inclusion involves meaningful participation by these students, achievement in accordance with their abilities, and social acceptance by teachers and peers. Teachers who view these students as a challenge rather than a burden, who believe that…

International Perspectives on Inclusion: Concluding Thoughts.

ERIC Educational Resources Information Center

Timmons, Vianne

2002-01-01

This article discusses overall trends and commonalities among previous articles on inclusion in the United Kingdom, Ireland, India, South Africa, Canada, New Zealand, and Australia. It concludes that inclusive education must be viewed in a broader context that looks at school reform and the societal context. Teacher education is stressed.…

State Early Childhood Inclusion Self-Assessment

ERIC Educational Resources Information Center

Whaley, Kathy T.; Cate, Debbie; Dell, Penny; Vinh, Megan; Neitzel, Jen

2017-01-01

This self-assessment tool provides a framework for examining key aspects of a State infrastructure that are useful for promoting high quality inclusive practices, programs and policies. The sections of the self-assessment are organized by the nine state recommendations of the "Policy Statement on Inclusion of Children with Disabilities in…

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

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

Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1more » vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V

Crossing the border - Solute entry into the chlamydial inclusion.

PubMed

Haferkamp, Ilka

2017-08-26

Chlamydiales comprise important human and animal pathogens as well as endosymbionts of amoebae. Generally, these obligate intracellular living bacteria are characterized by a biphasic developmental cycle, a reduced genome and a restricted metabolic capacity. Because of their metabolic impairment, Chlamydiales essentially rely on the uptake of diverse metabolites from their hosts. Chlamydiales thrive in a special compartment, the inclusion, and hence are surrounded by an additional membrane. Solutes might enter the inclusion through pores and open channels or by redirection of host vesicles, which fuse with the inclusion membrane and release their internal cargo. Recent investigations shed new light on the chlamydia-host interaction and identified an additional way for nutrient uptake into the inclusion. Proteome studies and targeting analyses identified chlamydial and host solute carriers in inclusions of Chlamydia trachomatis infected cells. These transporters are involved in the provision of UDP-glucose and biotin, and probably deliver further metabolites to the inclusion. By the controlled recruitment of specific solute carriers to the inclusion, the chlamydial resident thus can actively manipulate the metabolite availability and composition in the inclusion. This review summarizes recent findings and new ideas on carrier mediated solute uptake into the chlamydial inclusion in the context of the bacterial and host metabolism. Copyright © 2017 Elsevier GmbH. All rights reserved.

What Is Inclusive Didactics? Teachers' Understanding of Inclusive Didactics for Students with EBD in Swedish Mainstream Schools

ERIC Educational Resources Information Center

Gidlund, Ulrika; Boström, Lena

2017-01-01

Including students with emotional and behavioral difficulties (EBD) in general education is one of teachers' greatest challenges and make the dilemma of inclusion displays its most difficult side. This article contributes to the understanding of how teachers in Swedish mainstream schools understand the concept of inclusive didactics for students…

Can Teachers' Self-Reported Efficacy, Concerns, and Attitudes toward Inclusion Scores Predict Their Actual Inclusive Classroom Practices?

ERIC Educational Resources Information Center

Sharma, Umesh; Sokal, Laura

2016-01-01

This research was undertaken to determine if significant relationships exist between teachers' self-reported attitudes, concerns, and efficacy to teach in inclusive classrooms and their actual classroom behaviour in Winnipeg, Canada. Five teachers completed 3 scales measuring their attitudes to inclusion, their level of concerns about teaching in…

Developing Theories and Practices of Inclusion in Australia.

ERIC Educational Resources Information Center

Slee, Roger

2002-01-01

This article explores tensions between a general theory of inclusive education and those policies directed toward the schooling of students with disabilities. It examines theories of inclusive education, highlights tensions in the field of inclusive education, discusses these tensions as they are played out in Australia, and makes suggestions.…

Inclusion: The Pros and Cons--A Critical Review

ERIC Educational Resources Information Center

Savich, Carl

2008-01-01

The purpose of this paper was to review, analyze, and critique the pros and cons, the advantages and disadvantages, of inclusion. The methodology consisted in analyzing and comparing research findings on the benefits and costs of inclusion. Federal legislation and regulations on inclusion were examined, analyzed, and discussed. The results showed…

Communicative Approach to Inclusive Education in Pre-School

ERIC Educational Resources Information Center

Kraukle, Sandra

2015-01-01

The understanding of the basic principles of inclusive education motivates the inclusion of children with special needs in general education schools. The paper presents the process of implementing inclusive education in Latvia and the teachers' and parents' understanding of the role of communication in including children with special needs,…

An overview on current fluid-inclusion research and applications

USGS Publications Warehouse

Chi, G.; Chou, I.-Ming; Lu, H.-Z.

2003-01-01

This paper provides an overview of some of the more important developments in fluid-inclusion research and applications in recent years, including fluid-inclusion petrography, PVTX studies, and analytical techniques. In fluid-inclusion petrography, the introduction of the concept of 'fluid-inclusion assemblage' has been a major advance. In PVTX studies, the use of synthetic fluid inclusions and hydrothermal diamond-anvil cells has greatly contributed to the characterization of the phase behaviour of geologically relevant fluid systems. Various analytical methods are being developed and refined rapidly, with the Laser-Raman and LA-ICP-MS techniques being particularly useful for volatile and solute analyses, respectively. Ore deposit research has been and will continue to be the main field of application of fluid inclusions. However, fluid inclusions have been increasingly applied to other fields of earth science, especially in petroleum geology and the study of magmatic and earth interior processes.

Beyond Prejudice: Inclusive Learning in Practice

ERIC Educational Resources Information Center

Smith, Vikki; Armstrong, Anne

2005-01-01

Promoting an inclusive learning environment that caters for all learners and their individual needs and meeting challenging targets set in this area is a huge under taking for providers across the learning and skills sector. This booklet provides an overview that illustrates the breadth and variety that the broad banner of inclusive learning…

Inclusive Education: A Servant-Leadership Perspective

ERIC Educational Resources Information Center

Crippen, Carolyn

2005-01-01

The concept of inclusive education is common to the Canadian educational system and involves service to the students. According to Young and Levin, "The provision of appropriate education to meet the needs of each student is increasingly accepted as a right in Canadian education." Bloom, Perlmutter and Burnell define inclusion as "a…