| 1. |
- Alsterfjord, Magnus, et al.
(författare)
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Plasma membrane H+-ATPase and 14-3-3 Isoforms of Arabidopsis leaves: Evidence for isoform specificity in the 14-3-3/H+-ATPase interaction
- 2004
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 1471-9053 .- 0032-0781. ; 45:9, s. 1202-1210
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane H+-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H+-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H+-ATPase isoforms. We now show that the H'-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 istiforms tested bind to the H+-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H+-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H+-ATPase isoforms in leaves. However, mass peptide fingerprinting identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H+-ATPase. However, during a condition requiring full activation of H+ pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H+-ATPase.
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| 2. |
- Baureus Koch, Catrin, et al.
(författare)
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Interaction between weak low frequency magnetic fields and cell membranes.
- 2003
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Ingår i: Bioelectromagnetics. - : Wiley. - 0197-8462. ; 24:6, s. 395-402
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Tidskriftsartikel (refereegranskat)abstract
- The question of whether very weak low frequency magnetic fields can affect biological systems, has attracted attention by many research groups for quite some time. Still, today, the theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely low frequency (ELF) magnetic fields on the transport of Ca2+ was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested two quantum mechanical theoretical models that assume that biologically active ions can be bound to a channel protein and influence the opening state of the channel. Vesicles were exposed for 30 min at 32 °C and the calcium efflux was studied using radioactive 45Ca as a tracer. Static magnetic fields ranging from 27 to 37 T and time varying magnetic fields with frequencies between 7 and 72 Hz and amplitudes between 13 and 114 T (peak) were used. We show that suitable combinations of static and time varying magnetic fields directly interact with the Ca2+ channel protein in the cell membrane, and we could quantitatively confirm the model proposed by Blanchard. Bioelectromagnetics 24:395-402, 2003. © 2003 Wiley-Liss, Inc.
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| 3. |
- Christensen, Anna, et al.
(författare)
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Functional characterization of Arabidopsis calreticulin1a : a key alleviator of endoplasmic reticulum stress
- 2008
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Ingår i: Plant and Cell Physiology. - : Oxford University Press. - 0032-0781 .- 1471-9053. ; 49:6, s. 912-924
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Tidskriftsartikel (refereegranskat)abstract
- The chaperone calreticulin plays important roles in a variety of processes in the endoplasmic reticulum (ER) of animal cells, such as Ca2+ signaling and protein folding. Although the functions of calreticulin are well characterized in animals, only indirect evidence is available for plants. To increase our understanding of plant calreticulins we introduced one of the Arabidopsis isoforms, AtCRT1a, into calreticulin-deficient (crt–/–) mouse embryonic fibroblasts. As a result of calreticulin deficiency, the mouse crt–/– fibroblasts have decreased levels of Ca2+ in the ER and impaired protein folding abilities. Expression of the AtCRT1a in mouse crt–/– fibroblasts rescued these phenotypes, i.e. AtCRT1a restored the Ca2+-holding capacity and chaperone functions in the ER of the mouse crt–/– fibroblasts, demonstrating that the animal sorting machinery was also functional for a plant protein, and that basic calreticulin functions are conserved across the Kingdoms. Expression analyses using a β-glucuronidase (GUS)–AtCRT1a promoter construct revealed high expression of CRT1a in root tips, floral tissues and in association with vascular bundles. To assess the impact of AtCRT1a in planta, we generated Atcrt1a mutant plants. The Atcrt1a mutants exhibited increased sensitivity to the drug tunicamycin, an inducer of the unfolded protein response. We therefore conclude that AtCRT1a is an alleviator of the tunicamycin-induced unfolded protein response, and propose that the use of the mouse crt–/– fibroblasts as a calreticulin expression system may prove useful to assess functionalities of calreticulins from different species.
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| 4. |
- Christensen, Anna, et al.
(författare)
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Higher plant calreticulins have acquired specialized functions in arabidopsis
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:6, s. e11342-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Calreticulin (CRT) is a ubiquitous ER protein involved in multiple cellular processes in animals, such as protein folding and calcium homeostasis. Like in animals, plants have evolved divergent CRTs, but their physiological functions are less understood. Arabidopsis contains three CRT proteins, where the two CRTs AtCRT1a and CRT1b represent one subgroup, and AtCRT3 a divergent member. Methodology/Principal Findings: Through expression of single Arabidopsis family members in CRT-deficient mouse fibroblasts we show that both subgroups have retained basic CRT functions, including ER Ca2+-holding potential and putative chaperone capabilities. However, other more general cellular defects due to the absence of CRT in the fibroblasts, such as cell adhesion deficiencies, were not fully restored. Furthermore, in planta expression, protein localization and mutant analyses revealed that the three Arabidopsis CRTs have acquired specialized functions. The AtCRT1a and CRT1b family members appear to be components of a general ER chaperone network. In contrast, and as recently shown, AtCRT3 is associated with immune responses, and is essential for responsiveness to the bacterial Pathogen-Associated Molecular Pattern (PAMP) elf18, derived from elongation factor (EF)-Tu. Whereas constitutively expressed AtCRT1a fully complemented Atcrt1b mutants, AtCRT3 did not. Conclusions/Significance: We conclude that the physiological functions of the two CRT subgroups in Arabidopsis have diverged, resulting in a role for AtCRT3 in PAMP associated responses, and possibly more general chaperone functions for AtCRT1a and CRT1b.
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| 5. |
- Christensen, Anna, et al.
(författare)
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Overexpression of the Ca2+-binding protein calreticulin in the endoplasmic reticulum improves growth of tobacco cell suspensions (Nicotiana tabacum) in high-Ca2+ medium
- 2005
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 123:1, s. 92-99
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Tidskriftsartikel (refereegranskat)abstract
- Calreticulin (CRT) is a eukaryotic, highly conserved, Ca2+-binding protein predominantly located in the endoplasmic reticulum (ER) lumen. In addition to being involved in the regulation of cellular Ca2+, calreticulin is a key quality control element during protein folding in the ER lumen. Tobacco (Nicotiana tabacum L.) suspension cells overexpressing a maize CRT (CRT1a) were used here to examine the properties of CRT in growing plant cells with respect to stress exposure. The endogenous CRT gene was induced rapidly after subculturing of the cells to new medium. In accordance, the CRT protein levels increased, peaking at days 3-4. At day 5, when the CRT transcript levels had levelled off, a further increase in endogenous CRT expression was obtained when the cells were treated with excess Ca2+ or the N-linked glycosylation inhibitor tunicamycin. Whereas the response to Ca2+ occurred within 30 min, the induction by tunicamycin took several hours to be established. Transforming tobacco cells with maize CRT1a, under a constitutive mannopine synthase promoter, resulted in a stable level of expressed CRT1a during the growth cycle compared with endogenous CRT. The CRTs showed differences in attached glycans, but both contained the high mannose-rich-type glycans characteristic of ER proteins. In agreement with an ER location, both tobacco CRT and the transgene product CRT1a codistributed with the ER marker NADH cytochrome c reductase after density gradient centrifugation of microsomal fractions from tobacco cells. Increased production of CRT, as was obtained in the transgenic tobacco cell lines, made cells more tolerant than wild-type cells to high Ca2+ during growth. These data suggest that overexpression of CRT1a in plant cells results in a more efficient calcium buffering capacity in the ER.
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| 6. |
- Gomez, Federico, et al.
(författare)
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Biochemical aspects of carrot processing
- 2003
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Ingår i: International Symposium on Future Technologies for Food Production and Future Food Scientists, Proceedings. - 0280-9737. ; :162, s. 87-87
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Konferensbidrag (refereegranskat)
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| 7. |
- Gomez, Federico, et al.
(författare)
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Changes in the carrot (Daucus carota L. cv. Nerac) cell wall during storage
- 2004
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Ingår i: Food Research International. - : Elsevier BV. - 0963-9969. ; 37:3, s. 225-232
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to examine biochemical changes in cell wall carbohydrates and extensin proteins during long-term storage of carrots (Daucus carota L. cv. Nerac). During the storage period of 6 months, cell wall fractions were isolated from the carrot at various times for carbohydrate and protein analysis. Signs of extensin cross-linking and its concomitant insolubilisation in the cell wall were found after 7 and 12 weeks of storage. During the same period the concentration of galactose and arabinose decreased, while other carbohydrate components as well as the degree of methylesterification remained virtually unchanged. After the 12th week of storage no changes in the extensin or carbohydrates were detected. Oxidative cross-linking between extensin molecules in the cell wall has been implicated in cell wall strengthening and may be part of the mechanism behind the storage-induced firmness of carrots. (C) 2003 Elsevier Ltd. All rights reserved.
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| 8. |
- Gomez, Federico, et al.
(författare)
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Cold acclimation of carrots during storage mechanical properties and antifreezing protein.
- 2003
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Ingår i: Acta horticulturae : technical communications of ISHS. - 0567-7572. ; 599, s. 699-703
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Konferensbidrag (refereegranskat)abstract
- Changes in the composition of carrot cell wall proteins were investigated, associating metabolic changes during long-term storage with changes in mechanical properties. Harvested carrots accumulate an antifreezing protein in their cell walls reaching a maximum level after 12 weeks of storage at 0°C, followed by a gradual decrease. During the same period of time, there is a decrease in the slicing force during the first 7 weeks of storage followed by an increase until the 12th week. The appearance and accumulation of the antifreezing protein suggest that structural changes leading to changes in mechanical properties during the first 12 weeks of storage might be associated with a cold acclimation process.
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| 9. |
- Gomez, Federico, et al.
(författare)
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Influence of cold acclimation on the mechanical strength of carrot (Daucus carota L.) tissue
- 2004
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Ingår i: European Journal of Horticultural Science. - 1611-4434. ; 69:6, s. 229-234
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the influence of cold acclimation on the mechanical strength of carrot (Daucus carota L.) taproots. Changes in the mechanical strength were monitored when cold acclimation was induced in carrot plants cultivated in a growth chamber under strict climate control and in taproots harvested from field cultivation, where the plants had been exposed to the natural variations in climate. The appearance and accumulation of an antifreeze protein in the cell wall isolated from cold-stored taproots showed that a cold acclimation process is in progress in the harvested taproot derived from carrot plants grown in the field. The force needed to slice the taproots significantly increased during the first 12 weeks of storage, where the higher concentration of the antifreeze protein indicated the highest development of cold acclimation during that period of time. The increase in tissue rigidity during cold acclimation was also shown by the increase of the Young's modulus in taproot tissue from carrot plants acclimated 11 weeks under controlled temperature conditions. After 24 weeks of storage there was a significant increase in slicing force that was accompanied by signs of cell membrane deterioration, as measured by relative electrolyte leakage. Thus, the later increase in tissue strength might be related with a senescence process.
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| 10. |
- Gomez, Federico, et al.
(författare)
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On the induction of cold acclimation in carrots (Daucus carota L.) and its influence on storage performance
- 2005
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Ingår i: Food Research International. - : Elsevier BV. - 0963-9969. ; 38:1, s. 29-36
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the role of cold acclimation in carrot plants with respect to its influence on the storage performance of the harvested taproots. The induction of cold acclimation was followed in plants cultivated in a growth chamber under strict climate control and in taproots harvested from two separate field cultivations where the plants had been exposed to the natural variations in climate. Under controlled growth conditions, levels of antifreeze protein (AFP) mRNA were used as a marker for cold acclimation in carrot taproot tissue. Expression of this gene was induced by cold in discs excised from harvested taproots and this induction was clearly affected by the growth temperature of the plants from which the taproots were taken. These in vitro data were consistent with those from field-grown plants. In the cell wall of taproots harvested in year 2000, where the intact plants had frequently been exposed to temperatures below 6degreesC, a 36 kDa AFP accumulated to higher levels during storage than in the taproots harvested from plants grown in year 2001, where cultivation temperatures had rarely dropped below 6degreesC. The taproots from 2001 exhibited poor storage performance as shown by an earlier increase in relative electrolyte leakage and decrease in dry matter compared to taproots harvested in 2000. The capacity of the AFP to accumulate during storage was consistent with a high storage performance.
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| 11. |
- Gustavsson, Maria, et al.
(författare)
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Characterisation of a plasma membrane-associated phospholipase A(2) activity increased in response to cold acclimation
- 2002
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Ingår i: Journal of Plant Physiology. - : Elsevier BV. - 0176-1617. ; 159:11, s. 1219-1227
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Tidskriftsartikel (refereegranskat)abstract
- We here demonstrate the presence of a plasma membrane-associated phospholipase A(2) (EC 3.1.1.4; PLA(2)) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA(2) activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca2+, with full enzyme activity at 10mumol/L Ca2+. The Ca2+-dependent PLA(2) activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca2+-dependent PLA(2) activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA(2) activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA(2) in cold acclimation in plants.
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| 12. |
- Hunt, L, et al.
(författare)
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Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms
- 2004
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Ingår i: New Phytologist. - : Wiley. - 1469-8137 .- 0028-646X. ; 162:3, s. 643-654
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Tidskriftsartikel (refereegranskat)abstract
- PI-PLCs synthesise the calcium releasing second messenger IP3. We investigated the expression patterns of the Arabidopsis PI-PLC gene family and measured in vitro activity of encoded enzymes. Gene specific RT-PCR and promoter-GUS fusions were used to analyse AtPLC gene expression patterns. The five available AtPLC cDNAs were expressed as fusion proteins in Escherichia coli. All members of the AtPLC gene family were expressed in multiple organs of the plant. AtPLC1, and AtPLC5 expression was localized to the vascular cells of roots and leaves with AtPLC5::GUS also detected in the guard cells. AtPLC4::GUS was detected in pollen and cells of the stigma surface. In seedlings, AtPLC2 and AtPLC3 were constitutively expressed, while AtPLCs 1, 4 and 5 were induced by abiotic stresses. AtPLC1-5 were all shown to have phospholipase C activity in the presence of calcium ions. AtPLCs showed limited tissue specific expression and expression of at least three genes was increased by abiotic stress. The differing calcium sensitivities of recombinant AtPLC protein activities may provide a mechanism for generating calcium signatures.
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| 13. |
- Larsson, Christer, et al.
(författare)
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Plant Plasma membrane
- 2007
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Ingår i: Encyclopedia of Life Sciences. - : Wiley.
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Forskningsöversikt (refereegranskat)abstract
- The plasma membrane encloses the cell contents and acts as a barrier between the cell and its environment. The plasma membrane allows a controlled exchange of ions and solutes with the rest of the organism and its surroundings, and plays an active role in many processes, such as plant development, pathogen resistance and frost-hardiness.
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| 14. |
- Mikami, Koji, et al.
(författare)
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A dibasic amino acid pair conserved in the activation loop directs plasma membrane localization and is necessary for activity of plant type I/II Phosphatidylinositol Phosphate Kinase
- 2010
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 153:3, s. 1004-1015
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Tidskriftsartikel (refereegranskat)abstract
- Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.
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| 15. |
- Mikami, Koji, et al.
(författare)
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Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?
- 2010
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Ingår i: Plant Signalling & Behavior. - : Landes Bioscience. - 1559-2316 .- 1559-2324. ; 5:10, s. 1241-1244
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Tidskriftsartikel (refereegranskat)abstract
- Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol- 4,5-bisphosphate [PtdIns(4,5)P2], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs.
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| 16. |
- Olsson, Peter, et al.
(författare)
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Expression of bovine calmodulin in tobacco plants confers faster germination on saline media
- 2004
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Ingår i: Plant Science. - : Elsevier BV. - 0168-9452. ; 166:6, s. 1595-1604
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin is a Ca2+-dependent regulatory protein in eukaryotic cells involved in a variety of intracellular activities. Many responses to abiotic stresses involve signaling by Ca2+ and Ca2+/calmodulin regulation, including water and salinity stress. To investigate how calmodulin affects germination on media with high concentrations of NaCl, transgenic tobacco plants expressing heterologous calmodulin were generated and characterized. Transgenic tobacco seeds showed significantly shortened germination times on media containing varying salt (120-160 mM) and calcium (3 and 13 mM) concentrations, compared to control seeds. Using media containing 140 mM NaCl and 13 mM CaCl2, the average germination time was shortened by 2-3 days compared to control (P < 0.05). In addition, the germinating transgenic seeds contained higher transient levels of gamma-aminobutyric acid compared to control seeds (P < 0.05). The tobacco Ca2+/calmodulin-regulated glutamate decarboxylase, synthesizing gamma-aminobutyric acid may therefore be stimulated by the heterologous calmodulin. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
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| 17. |
- Ossipova, Elena, et al.
(författare)
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Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint
- 2014
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Ingår i: Arthritis Research & Therapy. - London : BioMed Central (BMC). - 1478-6362 .- 1478-6354. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint). METHODS: Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus(R) ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry. RESULTS: Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from alpha-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA. CONCLUSIONS: We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs. © 2014 Ossipova et al.; licensee BioMed Central Ltd.
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| 18. |
- Otterhag, L, et al.
(författare)
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Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase
- 2006
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Ingår i: Biochimie. - : Elsevier BV. - 1638-6183 .- 0300-9084. ; 88:1, s. 11-21
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Tidskriftsartikel (refereegranskat)abstract
- The A rabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1 expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp- 167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro tar et of AtPDK1. Our data clearly show that Asp- 167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident. (C) 2005 Elsevier SAS. All rights reserved.
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| 19. |
- Palmgren, Michael Gjedde, et al.
(författare)
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Sealed Inside-Out and Right-Side-Out Plasma Membrane Vesicles : Optimal Conditions for Formation and Separation
- 1990
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Ingår i: Plant Physiology. - 0032-0889 .- 1532-2548. ; 92:4, s. 871-880
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Tidskriftsartikel (refereegranskat)abstract
- Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H+ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H+ pumping were both completely inhibited by vanadate (Ki ≈ 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation.
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| 20. |
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| 21. |
- Persson, Staffan, et al.
(författare)
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Identification of a novel calreticulin isoform (Crt2) in human and mouse
- 2002
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Ingår i: Gene. - 1879-0038. ; 297:1-2, s. 151-158
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Tidskriftsartikel (refereegranskat)abstract
- Calreticulin is a Ca2+-binding chaperone localized mainly in the endoplasmic/sarcoplasmic reticulum in all higher organisms. To date, only one calreticulin isoform has been identified in human and mouse. Here we report a novel calreticulin isoform (Crt2) in human and mouse, with 53 (human) and 49% (mouse) identity to the previously identified calreticulin in respective species. The gene encoding the novel human calreticulin isoform spans 17 kb of genomic DNA and is expressed in testis, showing a similar expression as the chaperone calmegin. Phylogenetic analysis shows that two or more calreticulin (crt) genes are present both in plants and in mammals. The duplication of the crt gene in human and mouse suggests functional diversity, and variations in expression patterns among calreticulins. Two novel calreticulin (Crt2) isoforms, with high homology to the human and mouse calreticulin isoform (Crt2), were also identified in pig and rat via expressed sequence tags. (C) 2002 Elsevier Science B.V. All rights reserved.
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| 22. |
- Persson, Staffan, et al.
(författare)
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Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants.
- 2003
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 133:3, s. 1385-1396
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Tidskriftsartikel (refereegranskat)abstract
- Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.
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| 23. |
- Saavedra, Laura, et al.
(författare)
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Characterization of phosphatidylinositol phosphate kinases from the moss Physcomitrella patens : PpPIPK1 and PpPIPK2
- 2009
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Ingår i: Plant and Cell Physiology. - : Oxford University Press (OUP). - 0032-0781 .- 1471-9053. ; 50:3, s. 595-609
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoinositides (PIs) play a major role in eukaryotic cells, despite being a minor component of most membranes. This is the first report on PI metabolism in a bryophyte, the moss Physcomitrella patens. Moss PI composition is similar to that of other land plants growing under normal conditions. In contrast to the large number of PIPK genes present in flowering plants, the P. patens genome encodes only two type I/II PIPK genes, PpPIPK1 and PpPIPK2, which are very similar at both the nucleotide and protein product levels. However, the expression of the two genes is differentially regulated, and in vitro biochemical characterization shows that the resulting enzymes have different substrate specificities. PpPIPK1 uses PtdIns4P and PtdIns3P with similar preference and also metabolizes PtdIns(3,4)P(2) to produce PtdIns(3,4,5)P(3), a PI not yet detected in intact plant cells. PpPIPK2 prefers PtdIns as substrate and is much less active towards PtdIns4P and PtdIns3P. Thus, PpPIPK2 shows properties reminiscent of both PtdInsP-kinase and PtdIns-kinases. Moreover, a substitution of glutamic acid by alanine in the activation loop drastically reduced PpPIPK1 activity and altered the substrate specificity to PtdIns5P being the preferred substrate compared with PtdIns4P and PtdIns3P. These findings demonstrate that the substrate specificity of plant PIPKs is determined in a plant-specific manner, which provides new insights into the regulatory modes of PIPK activity in plants.
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| 24. |
- Saavedra, Laura, et al.
(författare)
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PIP kinases and their role in plant tip growing cells
- 2012
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Ingår i: Plant Signalling & Behavior. - : Taylor & Francis. - 1559-2316 .- 1559-2324. ; 7:10, s. 1302-1305
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Forskningsöversikt (refereegranskat)abstract
- Phosphatidylinositol (4,5) bisphosphate, [PtdIns(4,5)P2], is a signaling lipid involved in many important processes in animal cells such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels, and nuclear signaling pathways. In the last years PtdIns(4,5)P2 and its synthesizing enzyme, phosphatidylinositol phosphate kinase (PIPK), has been intensively studied in plant cells, revealing a key role in the control of polar tip growth. Analysis of the PIPK members from Arabidopsis thaliana, Oryza sativa and Physcomitrella patens showed that they share some regulatory features with animal PIPKs but also exert plant-specific modes of regulation. This review aims at giving an overview on the PIPK family from Arabidopsis thaliana and Physcomitrella patens. Even though their basic structure, modes of activation and physiological role is evolutionary conserved, modules responsible for plasma membrane localization are distinct for different PIPKs, depending on differences in physiological and/ or developmental status of cells, such as polarized and nonpolarized.
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| 25. |
- Saavedra, Laura, et al.
(författare)
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PIPKs are essential for rhizoid elongation and caulonemal cell development in the moss Physcomitrella patens
- 2011
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Ingår i: The Plant Journal. - 0960-7412 .- 1365-313X. ; 67:4, s. 635-647
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Tidskriftsartikel (refereegranskat)abstract
- PtdIns-4,5-bisphosphate is a lipid messenger of eukaryotic cells that plays a critical role in processes such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels and nuclear signalling pathways. The enzymes responsible for the synthesis of PtdIns(4,5)P(2) are phosphatidylinositol phosphate kinases (PIPKs). The moss Physcomitrella patens contains two PIPKs, PpPIPK1 and PpPIPK2. To study their physiological role, both genes were disrupted by targeted homologous recombination and as a result mutant plants with lower PtdIns(4,5)P(2) levels were obtained. A strong phenotype for pipk1, but not for pipk2 single knockout lines, was obtained. The pipk1 knockout lines were impaired in rhizoid and caulonemal cell elongation, whereas pipk1-2 double knockout lines showed dramatic defects in protonemal and gametophore morphology manifested by the absence of rapidly elongating caulonemal cells in the protonemal tissue, leafy gametophores with very short rhizoids, and loss of sporophyte production. pipk1 complemented by overexpression of PpPIPK1 fully restored the wild-type phenotype whereas overexpression of the inactive PpPIPK1E885A did not. Overexpression of PpPIPK2 in the pipk1-2 double knockout did not restore the wild-type phenotype demonstrating that PpPIPK1 and PpPIPK2 are not functionally redundant. In vivo imaging of the cytoskeleton network revealed that the shortened caulonemal cells in the pipk1 mutants was the result of the absence of the apicobasal gradient of cortical F-actin cables normally observed in wild-type caulonemal cells. Our data indicate that both PpPIPKs play a crucial role in the development of the moss P. patens, and particularly in the regulation of tip growth.
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| 26. |
- Salford, Leif, et al.
(författare)
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The mammalian brain in the electromagnetic fields designed by man with special reference to blood-brain barrier function, neuronal damage and possible physical mechanisms
- 2008
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Ingår i: PROGRESS OF THEORETICAL PHYSICS SUPPLEMENT. - 0375-9687. ; :173, s. 283-309
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Konferensbidrag (refereegranskat)abstract
- Life oil earth was formed during billions of years, exposed to, and shaped by the original physical forces such as gravitation, cosmic irradiation, atmospheric electric fields and the terrestrial magnetism. The Schumann resonances at 7.4 Hz are all example of oscillations possibly important for life.(1)) The existing organisms are created to function in harmony with these forces. However, in the late 19th century mankind introduced the use of electricity, in the early 20th century long-wave radio and in the 1940-ies short-wave radio. High frequency RF was introduced in the 50-ies as FM and television and during the very last decades, microwaves of the modern communication society spread around the world. Today, however, one third of the world's population is owner of the microwave-producing mobile phones and an even larger number is exposed to the cordless RF emitting systems. To what; extent are all living organisms affected by these, almost everywhere present radio frequency fields? And what will be the effects of many years of continuing exposure? Since 1989 Our group has studied the effects upon the mammalian blood-brain barrier (BBB) in rats by non-thermal radio frequency electromagnetic fields (RF-EMF). These have been shown to cause significantly increased leak-age of the rats' own blood albumin through the BBB of exposed rats, at energy levels of 1W/kg and below, as compared to non-exposed animals in a total series of about two thousand animals.(2)-6)) One remarkable observation is the fact that the lowest energy levels, with whole-body average power densities below 10mW/kg, give rise to the most pronounced albumin leakage. If mobile communication, even at extremely low energy levels, causes the users' own albumin to leak out through the BBB, also other unwanted and toxic molecules in the blood, may leak into the brain tissue and concentrate in and damage the neurons and glial cells of the brain. In later studies we have shown that a 2-h exposure to GSM 915 MHz, at non-thermal SAB-values of 0.2, 2 and 200 mW/kg, gives rise to significant neuronal damage, seen not only 50 days after the exposure 7) but also after 28 days but not after 14 days. Albumin extravasations and uptake into neurons was enhanced after 14 clays, but not after 28.(8)) in our continued research, also the non-thermal effects oil tissue structure and memory function of long-term exposure for 13 months are studied.(9)) We have also performed microarray analysis of brains from rats exposed to short term GSM both at 1,800 MHz and at 900MHz and have found significant effects upon gene expression of membrane associated genes as compared to control animals.(10),11)) Most of our findings support that living organisms are affected by the non-thermal radio frequency fields. Some other Studies agree while others find no effects. The mechanisms by which the EMFs may alter BBB permeability are not Well Understood. At low field strengths, the effects on body temperature are negligible and thus heating effects are not involved. A change in the physicochemical characteristics of membranes has been suggested as a cause.(12)) We have performed experiments to verify a quantum mechanical model for interaction with protein-bound ions. Our results show that controlled frequency and amplitude of ELF EM fields upon spinach plasma vesicles can steer transport over the membrane.(13)) This may be a first proof of a resonance phenomenon where appropriate levels of frequency and amplitude in the right combination have the potency to communicate with the biology of membranes and transport systems. Our study has prompted Lis to elaborate on magnetic resonance models; the Ion Cyclotron Resonance (ICR) model and the Ion Parametric Resonance (IPR) Model in an attempt to explain the occurrence of resonance frequencies. This is extensively described here under the heading: Mechanisms behind the effects of electromagnetical fields upon biology. We also bring forward the concept of solitons being active in membranes and DNA/RNA-transcription as a, possible mean to understand and prove the biological effects of EMF. The Nishinomiya-Yukawa International and Interdisciplinary Symposium 2007 raised the question: What is Life? An obvious and simple answer could be: It is DNA! The DNA strand can be looked upon as an antenna resonating in the microwave band 6GHz with its harmonics and subharmonics.(14)-18)) If this holds true, the dramatic situation might exist, that all living organisms have a receptor for the newly constructed and world-wide man-made microvaves, leading to a direct effect upon the function of DNA - in concordance with our experimental findings! Our generation invented the microwave emitters. We now have in imperative obligation to further investigate the links between EMF and biology in order to prevent possible detrimental effects of the microwaves.
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| 27. |
- Sehnke, PC, et al.
(författare)
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Evolution and isoform specificity of plant 14-3-3 proteins
- 2002
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Ingår i: Plant Molecular Biology. - 1573-5028. ; 50:6, s. 1011-1018
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Tidskriftsartikel (refereegranskat)abstract
- The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.
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| 28. |
- Sun, Wenjun J., et al.
(författare)
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Calcium efflux of plasma membrane vesicles exposed to ELF magnetic fieldsutest of a nuclear magnetic resonance interaction model
- 2012
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Ingår i: Bioelectromagnetics. - : Wiley. - 0197-8462. ; 33:7, s. 535-542
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Tidskriftsartikel (refereegranskat)abstract
- The question whether very weak, low frequency magnetic fields can affect biological matter is still under debate. The theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely weak 60?Hz magnetic fields on the transport of Ca2+ was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested a newly proposed quantum mechanical model postulates that polarization of hydrogen nuclei can elicit a biological effect. Vesicles were exposed for half an hour at 32?degrees C and the calcium efflux was studied using radioactive 45Ca2+ as a tracer. A static magnetic field of 26?mu T and time-varying magnetic fields with a frequency of 60?Hz and amplitudes between 0.6 and 6.3?mu T were used. The predictions of the model, proposed by Lednev, that at a frequency of 60?Hz the biological effect under investigation would significantly be altered at the amplitudes of 1.3 and 3.9?mu T could not be confirmed. Bioelectromagnetics 33:535542, 2012. (c) 2012 Wiley Periodicals, Inc.
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| 29. |
- Szilagyi, Anna, et al.
(författare)
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Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle - influence of alpha-tocopherol, cetylethers, linolenic acid, and temperature
- 2007
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 0006-3002 .- 1878-2434. ; 1768:9, s. 2310-2318
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Tidskriftsartikel (refereegranskat)abstract
- Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 'C, 25 'C and 37 'C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (Hit), such as linolenic acid the conversion increased, and the maximal level of violaxanthin deepoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L.), such as alpha-tocopherol and 8-cetylether (C16EO8), only 55% and 35% of the violaxanthin was converted at 25 degrees C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin deepoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase. (c) 2007 Elsevier B.V. All rights reserved.
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| 30. |
- Thelin, Lisa, et al.
(författare)
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Diverging functions among calreticulin isoforms in higher plants
- 2011
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Ingår i: Plant Signalling & Behavior. - : Taylor & Francis. - 1559-2316 .- 1559-2324. ; 6:6, s. 905-910
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The ER chaperone calreticulin plays vital roles in numerous cellular processes, including Ca2+-homeostasis, apoptosis and cell adhesion, in animal cells. Although calreticulin has been systematically characterized in animal cells, the focus has been on one of the isoforms. However, recent advances in the plant calreticulin field have revealed functional divergence of calreticulin isoforms. While two of the plant isoforms appear to work within a general ER chaperone framework, the third isoform is associated with folding of receptors for brassinosteroids and bacterial peptides. Hence, the discovery of functional specialization of plant calreticulins opens up new vistas for calreticulins also in the animal field.
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| 31. |
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| 32. |
- Wallstedt, Anna, et al.
(författare)
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The inhibition of ammonium uptake in excised birch (Betula pendula) roots by batatasin-III.
- 2001
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317. ; 113:3, s. 368-376
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Tidskriftsartikel (refereegranskat)abstract
- In northern Sweden, plants growing in association with the clonal dwarf shrub Empetrum hermaphroditum usually exhibit limited growth and are N-depleted. Previous studies suggest that this negative effect by E. hermaphroditum may be explained, at least in part, by the release of phenolic compounds, particularly the dihydrostilbene, batatasin-III from foliage to soil. In the present work, we investigated whether batatasin-III has the potential to interfere with NH4+ uptake in birch (Betula pendula) roots. Excised birch roots were exposed to batatasin-III during brief periods in 15NH4+ solutions, and then analyzed for labeled N. Batatasin-III inhibited N-NH4+ uptake by 28, 89 and 95% compared with the control, when roots were treated with 0.1, 1.0 and 2.8 mM of batatasin-III, respectively. The effect of 1.0-mM batatasin-III was greater at pH 4.2 than at pH 6.8. In addition, the inhibition of N-NH4+ uptake by batatasin-III was not reversed after rinsing the roots in water and transferring them to a batatasin-III free solution. Furthermore, birch seedlings immersed in a 1.0-mM batatasin-III solution for 2 h, and then replanted in pots with soil, had decreased growth, such that 10 weeks after treatment, the dry mass of both shoots and roots was reduced by 74 and 73%, respectively, compared with control seedlings. This suggests that a brief exposure to batatasin-III may have a long-term inhibitory effect on whole plant growth. Using plasma membrane vesicles isolated from easily extractable spinach (Spinacia oleracea) leaves, it was found that batatasin-III strongly inhibited proton pumping in isolated plasma membrane vesicles, while it only slightly inhibited ATP hydrolytic activity. The uncoupling of proton pumping from ATP hydrolytic activity suggests that batatasin-III disturbs membrane integrity. This hypothesis was further supported by a greater efflux of ions from birch roots immersed in a batatasin-III solution than from roots in a control solution.
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| 33. |
- Westergren, Tomas, et al.
(författare)
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Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms
- 1999
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Ingår i: Plant Physiology. - 1532-2548. ; 121:2, s. 507-516
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Tidskriftsartikel (refereegranskat)abstract
- Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent Km values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg2+ was the preferred divalent cation, Mn2+ could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn2+ acted synergistically with suboptimal Mg2+ concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca2+ and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of 3H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels.
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