Publications by authors named "Carlos Conde"

32 Publications

From the Nuclear Pore to the Fibrous Corona: A MAD Journey to Preserve Genome Stability.

Bioessays 2020 11 3;42(11):e2000132. Epub 2020 Sep 3.

i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, 4200-135, Portugal.

The relationship between kinetochores and nuclear pore complexes (NPCs) is intimate but poorly understood. Several NPC components and associated proteins are relocated to mitotic kinetochores to assist in different activities that ensure faithful chromosome segregation. Such is the case of the Mad1-c-Mad2 complex, the catalytic core of the spindle assembly checkpoint (SAC), a surveillance pathway that delays anaphase until all kinetochores are attached to spindle microtubules. Mad1-c-Mad2 is recruited to discrete domains of unattached kinetochores from where it promotes the rate-limiting step in the assembly of anaphase-inhibitory complexes. SAC proficiency further requires Mad1-c-Mad2 to be anchored at NPCs during interphase. However, the mechanistic relevance of this arrangement for SAC function remains ill-defined. Recent studies uncover the molecular underpinnings that coordinate the release of Mad1-c-Mad2 from NPCs with its prompt recruitment to kinetochores. Here, current knowledge on Mad1-c-Mad2 function and spatiotemporal regulation is reviewed and the critical questions that remain unanswered are highlighted.
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http://dx.doi.org/10.1002/bies.202000132DOI Listing
November 2020

VvERD6l13 is a grapevine sucrose transporter highly up-regulated in response to infection by Botrytis cinerea and Erysiphe necator.

Plant Physiol Biochem 2020 Sep 12;154:508-516. Epub 2020 Jul 12.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710-057, Braga, Portugal; Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro, Vila Real, Portugal.

The Early-Response to Dehydration six-like (ERD6l) is one of the largest families of sugar transporters in plants, however, is also one of the less studied with very few members characterized. In this work, we identified 18 members of the grapevine ERD6l family, analyzed their promoters and putative topology and additionally functionally characterized the member VvERD6l13. VvERD6l13 was strongly up-regulated in grape berries infected with Botrytis cinerea and Erysiphe necator in cv. Trincadeira and Carignan, respectively, suggesting an important role in grape berry-pathogen interaction, as we had hypothesized. In Cabernet Sauvignon Berry suspension cultured cells, VvERD6l13 was also up-regulated, by 4-fold, 48 h after elicitation with mycelium extract of B. cinerea. Besides being expressed in grape berries from various developmental stages, VvERD6l13 is also expressed in leaves, canes, flowers and, noticeably, in roots. Using tobacco and an hxt-null Saccharomyces cerevisiae strain as heterologous expression models, we showed that VvERD6l13 is localized at the plasma membrane and mediates the H-dependent transport of sucrose (K = 33 mM) thus confirming VvERD6l13 as a bona fide sugar transporter involved in sugar mobilization in grapevine and transcriptionally induced in response to biotic stress.
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http://dx.doi.org/10.1016/j.plaphy.2020.06.007DOI Listing
September 2020

The grapevine NIP2;1 aquaporin is a silicon channel.

J Exp Bot 2020 12;71(21):6789-6798

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.
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http://dx.doi.org/10.1093/jxb/eraa294DOI Listing
December 2020

RZZ-SPINDLY-DYNEIN: you got to keep 'em separated.

Cell Cycle 2020 07 16;19(14):1716-1726. Epub 2020 Jun 16.

IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto , Porto, Portugal.

To maintain genome stability, chromosomes must be equally distributed among daughter cells at the end of mitosis. The accuracy of chromosome segregation requires sister-kinetochores to stably attach to microtubules emanating from opposite spindle poles. However, initial kinetochore-microtubule interactions are able to turnover so that defective attachment configurations that typically arise during early mitosis may be corrected. Growing evidence supports a role for the RZZ complex in preventing the stabilization of erroneous kinetochore-microtubule attachments. This inhibitory function of RZZ toward end-on attachments is relieved by DYNEIN-mediated transport of the complex as chromosomes congress and appropriate interactions with microtubules are established. However, it remains unclear how DYNEIN is antagonized to prevent premature RZZ removal. We recently described a new mechanism that sheds new light on this matter. We found that POLO kinase phosphorylates the DYNEIN adaptor SPINDLY to promote the uncoupling between RZZ and DYNEIN. Elevated POLO activity during prometaphase ensures that RZZ is retained at kinetochores to allow the dynamic turnover of kinetochore-microtubule interactions and prevent the stabilization of erroneous attachments. Here, we discuss additional interpretations to explain a model for POLO-dependent regulation of the RZZ-SPINDLY-DYNEIN module during mitosis.
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http://dx.doi.org/10.1080/15384101.2020.1780382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469663PMC
July 2020

Spindle checkpoint: trapped by the corona, cyclin B1 goes MAD.

EMBO J 2020 06 18;39(12):e105279. Epub 2020 May 18.

Instituto de Investigação e Inovação em Saúde-i3S, Universidade do Porto, Porto, Portugal.

The spindle checkpoint protects against aneuploidy by ensuring that dividing cells only proceed with chromosome segregation once all kinetochores are stably attached to spindle microtubules. The checkpoint protein MAD1 localizes to the corona, a structural expansion of the kinetochore forming in the absence of microtubule attachment, but molecular mechanism or functional significance of this localization remains unknown. Recent results now show that cyclin B1 recruits MAD1 to the corona and that this MAD1 pool is required for robust checkpoint signaling.
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http://dx.doi.org/10.15252/embj.2020105279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298284PMC
June 2020

Sweet Cherry ( L.) PaPIP1;4 Is a Functional Aquaporin Upregulated by Pre-Harvest Calcium Treatments that Prevent Cracking.

Int J Mol Sci 2020 Apr 24;21(8). Epub 2020 Apr 24.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, Universidade do Minho, 4710-057 Braga, Portugal.

The involvement of aquaporins in rain-induced sweet cherry ( L.) fruit cracking is an important research topic with potential agricultural applications. In the present study, we performed the functional characterization of PaPIP1;4, the most expressed aquaporin in sweet cherry fruit. Field experiments focused on the pre-harvest exogenous application to sweet cherry trees, cultivar Skeena, with a solution of 0.5% CaCl, which is the most common treatment to prevent cracking. Results show that PaPIP1;4 was mostly expressed in the fruit peduncle, but its steady-state transcript levels were higher in fruits from CaCl-treated plants than in controls. The transient expression of PaPIP1;4-GFP in tobacco epidermal cells and the overexpression of PaPIP1;4 in YSH1172 yeast mutation showed that PaPIP1;4 is a plasma membrane protein able to transport water and hydrogen peroxide. In this study, we characterized for the first time a plasma membrane sweet cherry aquaporin able to transport water and HO that is upregulated by the pre-harvest exogenous application of CaCl supplements.
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http://dx.doi.org/10.3390/ijms21083017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215675PMC
April 2020

VvSWEET7 Is a Mono- and Disaccharide Transporter Up-Regulated in Response to Infection in Grape Berries.

Front Plant Sci 2019 27;10:1753. Epub 2020 Jan 27.

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Braga, Portugal.

The newly-identified SWEETs are high-capacity, low-affinity sugar transporters with important roles in numerous physiological mechanisms where sugar efflux is critical. SWEETs are desirable targets for manipulation by pathogens and their expression may be transcriptionally reprogrammed during infection. So far, few plant SWEET transporters have been functionally characterized, especially in grapevine. In this study, in the -susceptible variety "Trincadeira," we thoroughly analyzed modifications in the gene expression profile of key genes in -infected grape berries. VvSWEET7 and VvSWEET15 are likely to play an important role during fruit development and infection as they are strongly expressed at the green and mature stage, respectively, and were clearly up-regulated in response to infection. Also, infection down-regulated expression at the green stage, and expression at the veraison stage, and expression at the mature stage. VvSWEET7 was functionally characterized by heterologous expression in as a low-affinity, high-capacity glucose and sucrose transporter with a of 15.42 mM for glucose and a of 40.08 mM for sucrose. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in epidermal cells and confocal microscopy allowed to observe that both proteins clearly localize to the plasma membrane. In sum, VvSWEETs transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to infection.
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http://dx.doi.org/10.3389/fpls.2019.01753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996298PMC
January 2020

Mps1-mediated release of Mad1 from nuclear pores ensures the fidelity of chromosome segregation.

J Cell Biol 2020 03;219(3)

i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

The spindle assembly checkpoint (SAC) relies on the recruitment of Mad1-C-Mad2 to unattached kinetochores but also on its binding to Megator/Tpr at nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis remain elusive. Here, we show that activation of Mps1 during prophase triggers Mad1 release from NPCs and that this is required for kinetochore localization of Mad1-C-Mad2 and robust SAC signaling. We find that Mps1 phosphorylates Megator/Tpr to reduce its interaction with Mad1 in vitro and in Drosophila cells. Importantly, preventing Mad1 from binding to Megator/Tpr restores Mad1 accumulation at kinetochores, the fidelity of chromosome segregation, and genome stability in larval neuroblasts of mps1-null mutants. Our findings demonstrate that the subcellular localization of Mad1 is tightly coordinated with cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently preserve genomic stability.
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http://dx.doi.org/10.1083/jcb.201906039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054998PMC
March 2020

Polo regulates Spindly to prevent premature stabilization of kinetochore-microtubule attachments.

EMBO J 2020 01 18;39(2):e100789. Epub 2019 Dec 18.

IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.
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http://dx.doi.org/10.15252/embj.2018100789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960449PMC
January 2020

α-Fodrin is required for the organization of functional microtubules during mitosis.

Cell Cycle 2019 Oct 27;18(20):2713-2726. Epub 2019 Aug 27.

Cancer Research Program-III, Rajiv Gandhi Centre for Biotechnology, University of Kerala , Thiruvananthapuram , India.

The cytoskeleton protein α-fodrin plays a major role in maintaining structural stability of membranes. It was also identified as part of the brain γ-tubulin ring complex, the major microtubule nucleator. Here, we investigated the requirement of α-fodrin for microtubule spindle assembly during mitotic progression. We found that α-fodrin depletion results in abnormal mitosis with uncongressed chromosomes, leading to prolonged activation of the spindle assembly checkpoint and a severe mitotic delay. Further, α-fodrin repression led to the formation of shortened spindles with unstable kinetochore-microtubule attachments. We also found that the mitotic kinesin CENP-E had reduced levels at kinetochores to likely account for the chromosome misalignment defects in α-fodrin-depleted cells. Importantly, we showed these cells to exhibit reduced levels of detyrosinated α-tubulin, which primarily drives CENP-E localization. Since proper microtubule dynamics and chromosome alignment are required for completion of normal mitosis, this study reveals an unforeseen role of α-fodrin in regulating mitotic progression. Future studies on these lines of observations should reveal important mechanistic insight for fodrin's involvement in cancer.
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http://dx.doi.org/10.1080/15384101.2019.1656476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773225PMC
October 2019

Cell Cycle Kinase Polo Is Controlled by a Widespread 3' Untranslated Region Regulatory Sequence in Drosophila melanogaster.

Mol Cell Biol 2019 08 16;39(15). Epub 2019 Jul 16.

Gene Regulation, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

Alternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3' untranslated region (3'UTR), with critical physiological consequences. Here, we characterized the molecular mechanisms required for alternative polyadenylation. We identified a conserved upstream sequence element (USE) close to the proximal poly(A) signal. Transgenic flies without this sequence show incorrect selection of poly(A) signals with consequent downregulation of Polo expression levels and insufficient/defective activation of Polo kinetochore targets Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequence We found that Hephaestus binds to the USE RNA and that mutants display defects in alternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of noncanonical polyadenylation signals in genes. Taken together, our results revealed the molecular mechanisms involved in alternative polyadenylation, with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a new function for USEs in .
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http://dx.doi.org/10.1128/MCB.00581-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639250PMC
August 2019

Phosphatases in Mitosis: Roles and Regulation.

Biomolecules 2019 02 7;9(2). Epub 2019 Feb 7.

IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal.

Mitosis requires extensive rearrangement of cellular architecture and of subcellular structures so that replicated chromosomes can bind correctly to spindle microtubules and segregate towards opposite poles. This process originates two new daughter nuclei with equal genetic content and relies on highly-dynamic and tightly regulated phosphorylation of numerous cell cycle proteins. A burst in protein phosphorylation orchestrated by several conserved kinases occurs as cells go into and progress through mitosis. The opposing dephosphorylation events are catalyzed by a small set of protein phosphatases, whose importance for the accuracy of mitosis is becoming increasingly appreciated. This review will focus on the established and emerging roles of mitotic phosphatases, describe their structural and biochemical properties, and discuss recent advances in understanding the regulation of phosphatase activity and function.
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http://dx.doi.org/10.3390/biom9020055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406801PMC
February 2019

The grapevine VvCAX3 is a cation/H exchanger involved in vacuolar Ca homeostasis.

Planta 2017 Dec 11;246(6):1083-1096. Epub 2017 Aug 11.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas, CITAB-UMinho Pole, Departamento de Biologia, Escola de Ciências, Universidade do Minho, Braga, Portugal.

Main Conclusion: The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca , Na and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca, demonstrating a role in Ca transport. The truncated VvCAX3 was further shown to be involved in the transport of Na, Li, Mn and Cu in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca, and Na in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.
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http://dx.doi.org/10.1007/s00425-017-2754-0DOI Listing
December 2017

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

Elife 2017 05 2;6. Epub 2017 May 2.

i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show and in that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.
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http://dx.doi.org/10.7554/eLife.25366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433843PMC
May 2017

The Grapevine Uncharacterized Intrinsic Protein 1 (VvXIP1) Is Regulated by Drought Stress and Transports Glycerol, Hydrogen Peroxide, Heavy Metals but Not Water.

PLoS One 2016 9;11(8):e0160976. Epub 2016 Aug 9.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas CITAB, Vila Real, Portugal.

A MIP (Major Intrinsic Protein) subfamily called Uncharacterized Intrinsic Proteins (XIP) was recently described in several fungi and eudicot plants. In this work, we cloned a XIP from grapevine, VvXIP1, and agrobacterium-mediated transformation studies in Nicotiana benthamiana revealed that the encoded aquaporin shows a preferential localization at the endoplasmic reticulum membrane. Stopped-flow spectrometry in vesicles from the aqy-null yeast strain YSH1172 overexpressing VvXIP1 showed that VvXIP1 is unable to transport water but is permeable to glycerol. Functional studies with the ROS sensitive probe CM-H2DCFDA in intact transformed yeasts showed that VvXIP1 is also able to permeate hydrogen peroxide (H2O2). Drop test growth assays showed that besides glycerol and H2O2, VvXIP1 also transports boric acid, copper, arsenic and nickel. Furthermore, we found that VvXIP1 transcripts were abundant in grapevine leaves from field grown plants and strongly repressed after the imposition of severe water-deficit conditions in potted vines. The observed downregulation of VvXIP1 expression in cultured grape cells in response to ABA and salt, together with the increased sensitivity to osmotic stress displayed by the aqy-null yeast overexpressing VvXIP1, corroborates the role of VvXIP1 in osmotic regulation besides its involvement in H2O2 transport and metal homeostasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978503PMC
August 2017

Autoimmune post-herpes simplex encephalitis of adults and teenagers.

Neurology 2015 Nov 21;85(20):1736-43. Epub 2015 Oct 21.

From the Neuroimmunology Program (T.A., S.L., M.R., F.G., J.D.), August Pi Sunyer Biomedical Research Institute (IDIBAPS), and the Department of Neurology (S.L., F.G), Hospital Clínic, University of Barcelona; the Department of Neurology (G.M.) and the Pediatric Neurology Unit, Pediatrics Department (I.M.), Hospital Universitario Central de Asturias, Oviedo; the Department of Pediatric Neurology (V.C.-E., L.G.-G.-S.), Hospital Universitario Niño Jesús, Madrid, Spain; the Department of Neurology (C.E.C., G.G.), Hospital Universitario Hernando Moncaleano Perdomo, Neiva, Colombia; the Department of Pediatric Neurology (K.R.), Children's Hospital Datteln, Witten/Herdecke University, Witten, Germany; the Department of Neurology (M.E.E., I.C.-N.), Complejo Hospitalario de Navarra, Pamplona; the Department of Neurology (J.C.P.-C.), Hospital San Pedro de Alcántara, Cáceres; the Pediatric Neurology Unit (E.T.-V.), Hospital de la Santa Creu i Sant Pau, Barcelona; the Pediatric Neurology Unit (B.M.-C.), Hospital Universitario Virgen del Rocio, Sevilla; the Department of Pediatric Neurology (C.T.-T.), Hospital General La Mancha Centro, Alcázar de San Juan, Spain; the Department of Neurology (J.D.), University of Pennsylvania, Philadelphia; and the Catalan Institution for Research and Advanced Studies (ICREA) (J.D.), Barcelona, Spain.

Objective: To report 14 patients with immune-mediated relapsing symptoms post-herpes simplex encephalitis (HSE) and to compare the clinical and immunologic features of the teenage and adult group with those of young children.

Methods: Prospective observational study of patients diagnosed between June 2013 and February 2015. Immunologic techniques have been reported previously.

Results: Among the teenage and adult group (8 patients, median age 40 years, range 13-69; 5 male), 3 had an acute symptom presentation suggesting a viral relapse, and 5 a presentation contiguous with HSE suggesting a recrudescence of previous deficits. Seven patients developed severe psychiatric/behavioral symptoms disrupting all social interactions, and one refractory status epilepticus. Blepharospasm occurred in one patient. Five patients had CSF antibodies against NMDA receptor (NMDAR) and 3 against unknown neuronal cell surface proteins. In 5/6 patients, the brain MRI showed new areas of contrast enhancement that decreased after immunotherapy and clinical improvement. Immunotherapy was useful in 7/7 patients, sometimes with impressive recoveries, returning to their baseline HSE residual deficits. Compared with the 6 younger children (median age 13 months, range 6-20, all with NMDAR antibodies), the teenagers and adults were less likely to develop choreoathetosis (0/8 vs 6/6, p < 0.01) and decreased level of consciousness (2/8 vs 6/6, p < 0.01) and had longer delays in diagnosis and treatment (interval relapse/antibody testing 85 days, range 17-296, vs 4 days, range 0-33, p = 0.037).

Conclusion: In teenagers and adults, the immune-mediated relapsing syndrome post-HSE is different from that known in young children as choreoathetosis post-HSE and is underrecognized. Prompt diagnosis is important because immunotherapy can be highly effective.
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http://dx.doi.org/10.1212/WNL.0000000000002125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653102PMC
November 2015

Identification and functional characterization of grapevine transporters that mediate glucose-6-phosphate uptake into plastids.

Planta 2015 Oct 26;242(4):909-20. Epub 2015 May 26.

Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas CITAB, Vila Real, Portugal.

Main Conclusion: Two grapevine glucose-6-Pi plastidial transporters differently expressed in plant organs and in response to environmental and hormonal signals are characterized. They are involved in starch accumulation in berries and canes. In grapevine, starch accumulation in the trunk is important for winter storage of carbon and in the flower for reproductive development. Berries also accumulate starch in their plastids, which are also involved in the synthesis of aroma compounds important for fruit quality. The present work characterizes two glucose-phosphate translocators (VvGPT1, VvGPT2) that control the accumulation of starch in grape amyloplasts. Three different splicing variants identified for VvGPT2 (VvGPT2α, VvGPT2β and VvGPT2Ω) were more expressed in the leaves than in other organs. In contrast, VvGPT1 transcripts were more abundant in mature berries, canes and flowers than in the leaves. Expression of 35S-VvGPT1-GFP and 35S-VvGPT2Ω-GFP in tobacco leaf epidermal cells showed that the fusion proteins localized at the plastidial envelope. Complementation of the Arabidopsis pgi1-1 mutant impaired in leaf starch synthesis restored its ability to synthesize starch, demonstrating that VvGPT1 and VvGPT2Ω mediate the transport of glucose-6-Pi across the plastidial envelope. In grape cell suspensions, ABA, light and galactinol, together with sucrose and fructose, significantly increased the transcript abundance of VvGPT1, whereas VvGPT2Ω expression was affected only by sucrose. In addition, elicitation with methyl jasmonate strongly upregulated VvGPT1, VvGPT2Ω and VvPAL1, suggesting a role for GPTs in the production of secondary compounds in grapevine. Moreover, in grapevines cultivated in field conditions, VvGPT1 expression was higher in berries more exposed to the sun and subjected to higher temperatures. Although both VvGPT1 and VvGPT2 mediate the same function at the molecular level, they exhibit different expression levels and regulation in plant organs and in response to environmental and hormonal signals.
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http://dx.doi.org/10.1007/s00425-015-2329-xDOI Listing
October 2015

All together now: Polo joins the kinase network controlling the spindle assembly checkpoint in Drosophila.

Fly (Austin) 2013 Oct-Dec;7(4):224-8. Epub 2013 Aug 29.

IBMC; Instituto de Biologia Molecular e Celular; Universidade do Porto; Porto, Portugal; ICBAS; Department of Molecular Biology; Instituto de Ciências Biomédicas Abel Salazar; Universidade do Porto; Porto, Portugal.

Maintenance of genomic stability during eukaryotic cell division relies on the Spindle Assembly Checkpoint (SAC), which has evolved as a surveillance mechanism that monitors kinetochore-microtubule attachment and prevents APC/C-mediated mitotic exit until all chromosomes are properly attached to the mitotic spindle. Reversible protein phosphorylation has long been accredited as a regulatory mechanism of the SAC. Nevertheless, knowledge of how several mitotic kinases act in concert within the signaling pathway to orchestrate SAC function is still emerging. In a recent study, we undertook a comprehensive dissection of the hierarchical framework controlling SAC function in Drosophila cells. We found that Polo lies at the top of the SAC pathway promoting the efficient recruitment of Mps1 to unattached kinetochores. This renders Mps1 fully active to control BubR1 phosphorylation that generates the 3F3/2 phosphoepitope at tensionless kinetochores. We have proposed that Polo is required for SAC function and that the molecular outcome of Mps1-dependent 3F3/2 formation is to promote the association of Cdc20 with BubR1 allowing proper kinetochore recruitment of Cdc20 and efficient assembly of the Mitotic Checkpoint Complex (MCC) required for a sustained SAC response.
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http://dx.doi.org/10.4161/fly.26231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896493PMC
May 2015

Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation.

EMBO J 2013 Jun 17;32(12):1761-77. Epub 2013 May 17.

Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.
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http://dx.doi.org/10.1038/emboj.2013.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680734PMC
June 2013

Assessing and forecasting the impacts of global change on Mediterranean rivers. The SCARCE Consolider project on Iberian basins.

Environ Sci Pollut Res Int 2012 May 29;19(4):918-33. Epub 2012 Apr 29.

IDAEA-CSIC, Jordi Girona, 18-26, 08034, Barcelona, Spain.

Introduction: The Consolider-Ingenio 2010 project SCARCE, with the full title "Assessing and predicting effects on water quantity and quality in Iberian Rivers caused by global change" aims to examine and predict the relevance of global change on water availability, water quality, and ecosystem services in Mediterranean river basins of the Iberian Peninsula, as well as their socio-economic impacts. Starting in December 2009, it brought together a multidisciplinary team of 11 partner Spanish institutions, as well as the active involvement of water authorities, river basin managers, and other relevant agents as stakeholders.

Methods: The study areas are the Llobregat, Ebro, Jucar, and Guadalquivir river basins. These basins have been included in previous studies and projects, the majority of whom considered some of the aspects included in SCARCE but individually. Historical data will be used as a starting point of the project but also to obtain longer time series. The main added value of SCARCE project is the inclusion of scientific disciplines ranging from hydrology, geomorphology, ecology, chemistry, and ecotoxicology, to engineering, modeling, and economy, in an unprecedented effort in the Mediterranean area. The project performs data mining, field, and lab research as well as modeling and upscaling of the findings to apply them to the entire river basin.

Results: Scales ranging from the laboratory to river basins are addressed with the potential to help improve river basin management. The project emphasizes, thus, linking basic research and management practices in a single framework. In fact, one of the main objectives of SCARCE is to act as a bridge between the scientific and the management and to transform research results on management keys and tools for improving the River Basin Management Plans. Here, we outline the general structure of the project and the activities conducted within the ten Work Packages of SCARCE.
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http://dx.doi.org/10.1007/s11356-011-0566-5DOI Listing
May 2012

POLO ensures chromosome bi-orientation by preventing and correcting erroneous chromosome-spindle attachments.

J Cell Sci 2012 Feb;125(Pt 3):576-83

IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal.

Correct chromosome segregation during cell division requires bi-orientation at the mitotic spindle. Cells possess mechanisms to prevent and correct inappropriate chromosome attachment. Sister kinetochores assume a 'back-to-back' geometry on chromosomes that favors amphitelic orientation but the regulation of this process and molecular components are unknown. Abnormal chromosome-spindle interactions do occur but are corrected through the activity of Aurora B, which destabilizes erroneous attachments. Here, we address the role of Drosophila POLO in chromosome-spindle interactions and show that, unlike inhibition of its activity, depletion of the protein results in bipolar spindles with most chromosomes forming stable attachments with both sister kinetochores bound to microtubules from the same pole in a syntelic orientation. This is partly the result of impaired localization and activity of Aurora B but also of an altered centromere organization with abnormal distribution of centromeric proteins and shorter interkinetochore distances. Our results suggests that POLO is required to promote amphitelic attachment and chromosome bi-orientation by regulating both the activity of the correction mechanism and the architecture of the centromere.
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http://dx.doi.org/10.1242/jcs.092445DOI Listing
February 2012

Chromosomal localisation of five genes in Perkinsus olseni (Phylum Perkinsozoa).

Eur J Protistol 2012 Aug 17;48(3):194-8. Epub 2012 Feb 17.

Molecular Genetic Laboratory, Institute of Biomedical Sciences Abel Salazar, University of Porto, P-4099-003 Porto, Portugal.

The molecular karyotype of Perkinsus olseni, a pathogenic protist that infects the clam Ruditapes decussatus, comprises nine chromosomes, ranging in size from 0.15 Mb to 6.5 Mb, representing a haploid genome of about 28 Mb. In order to establish chromosome specific markers, PCR-amplified DNA sequences belonging to five conserved genes (18S rRNA, actin type I, hsp90, β-tubulin and calmodulin) were hybridised to chromosomal bands separated by pulsed-field gel electrophoresis. Three of those probes (actin type I, hsp90 and calmodulin) hybridised to only one chromosome and the remaining two (18S rRNA and β-tubulin) hybridised to two chromosomes. In the first place, the hybridisation pattern obtained serves to dispel any doubt about the nuclear location of the smallest chromosome observed in the molecular karyotype of Perkinsus olseni. Additionally, it will be a reference for further analysis of karyotype polymorphisms in the genus Perkinsus.
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http://dx.doi.org/10.1016/j.ejop.2011.11.002DOI Listing
August 2012

Mannitol transport and mannitol dehydrogenase activities are coordinated in Olea europaea under salt and osmotic stresses.

Plant Cell Physiol 2011 Oct 4;52(10):1766-75. Epub 2011 Sep 4.

Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB), Portugal.

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.
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http://dx.doi.org/10.1093/pcp/pcr121DOI Listing
October 2011

Emotion processing for arousal and neutral content in Alzheimer's disease.

Int J Alzheimers Dis 2010 Feb 1;2009. Epub 2010 Feb 1.

Laboratory of Neurosciences and Behavior, Department of Physiological Sciences, Institute of Biology, University of Brasilia, Brasilia-DF CEP 70910-900, Brazil.

Objective. To assess the ability of Alzheimer's disease (AD) patients to perceive emotional information and to assign subjective emotional rating scores to audiovisual presentations. Materials and Methods. 24 subjects (14 with AD, matched to controls for age and educational levels) were studied. After neuropsychological assessment, they watched a Neutral story and then a story with Emotional content. Results. Recall scores for both stories were significantly lower in AD (Neutral and Emotional: P = .001). CG assigned different emotional scores for each version of the test, P = .001, while ratings of AD did not differ, P = .32. Linear regression analyses determined the best predictors of emotional rating and recognition memory for each group among neuropsychological tests battery. Conclusions. AD patients show changes in emotional processing on declarative memory and a preserved ability to express emotions in face of arousal content. The present findings suggest that these impairments are due to general cognitive decline.
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http://dx.doi.org/10.4061/2009/278615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2915644PMC
February 2010

Physiological, biochemical and molecular changes occurring during olive development and ripening.

J Plant Physiol 2008 Oct 20;165(15):1545-62. Epub 2008 Jun 20.

Departamento de Biologia, Universidade do Minho, Campus de Gualtar, Braga, Portugal.

Since ancient times the olive tree (Olea europaea), an evergreen drought- and moderately salt-tolerant species, has been cultivated for its oil and fruit in the Mediterranean basin. Olive is unique among the commercial important oil crops for many reasons. Today, it ranks sixth in the world's production of vegetable oils. Due to its nutritional quality, olive oil has a high commercial value compared with most other plant oils. Olive oil has a well-balanced composition of fatty acids, with small amounts of palmitate, and it is highly enriched in the moneonic acid oleate. This makes it both fairly stable against auto-oxidation and suitable for human health. Nevertheless, it is the presence of minor components, in particular phenolics, contributing for oil's high oxidative stability, color and flavor, that makes olive oil unique among other oils. Moreover, as a result of their demonstrated roles in the prevention of cancer and cardiovascular diseases, olive phenolics have gained much attention during the past years. Also unique to virgin olive oil is its characteristic aroma. This results from the formation of volatile compounds, namely, aldehydes and alcohols of six carbon atoms, which is triggered when olives are crushed during the process of oil extraction. The biochemistry of the olive tree is also singular. O. europaea is one of the few species able to synthesize both polyols (mannitol) and oligosaccharides (raffinose and stachyose) as the final products of the photosynthetic CO(2) fixation in the leaf. These carbohydrates, together with sucrose, can be exported from leaves to fruits to fulfill cellular metabolic requirements and act as precursors to oil synthesis. Additionally, developing olives contain active chloroplasts capable of fixing CO(2) and thus contributing to the carbon economy of the fruit. The overall quality of table olives and olive oil is influenced by the fruit ripening stage. Olive fruit ripening is a combination of physiological and biochemical changes influenced by several environmental and cultural conditions, even if most events are under strict genetic control.
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http://dx.doi.org/10.1016/j.jplph.2008.04.018DOI Listing
October 2008

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells.

Biochim Biophys Acta 2007 Nov 25;1768(11):2801-11. Epub 2007 Jul 25.

Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.
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http://dx.doi.org/10.1016/j.bbamem.2007.07.010DOI Listing
November 2007

OeMST2 encodes a monosaccharide transporter expressed throughout olive fruit maturation.

Plant Cell Physiol 2007 Sep 27;48(9):1299-308. Epub 2007 Jul 27.

Departamento de Biologia, Universidade do Minho, Campus de Gualtar 4710-057, Braga, Portugal.

In olive fruits, sugars are the main soluble components providing energy and acting as precursors for olive oil biosynthesis. Large quantities of glucose, fructose and galactose are often found in olive pulp. To analyze sugar transport processes in Olea europaea, a cDNA encoding a monosaccharide transporter, designated OeMST2 (Olea europaea monosaccharide transporter 2) was cloned. An open reading frame of 1,569 bp codes for a protein of 523 amino acids and a calculated molecular weight of 57.6 kDa. The protein is homologous to other sugar transporters identified so far in higher plants. Expression of this cDNA in an hxt-null Saccharomyces cerevisiae strain deficient in glucose transport restored its capacity to grow on and to transport glucose. The encoded protein showed high affinity for D-glucose (K(m), 25 microM) and was also able to recognize D-galactose and the analogs 3-O-methyl-D-glucose and 2-deoxy-D-glucose, but not D-fructose, D-arabinose, sucrose or D-mannitol. Maximal transport activity was high at acidic pH (5.0), and the initial D-[(14)C]glucose uptake rates were strongly inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, confirming that OeMST2 is a H(+)/monosaccharide transporter. The expression of OeMST2 was studied during the ripening process. Transcript levels increased during fruit maturation, suggesting that OeMST2 takes part in the massive accumulation of monosaccharides in olive fruits. Monosaccharide:H(+) transport system activity and OeMST2 expression were negatively regulated by glucose in suspension-cultured cells. Glucose-mediated OeMST2 repression was impaired by mannoheptulose, suggesting the involvement of a hexokinase-dependent signaling pathway.
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http://dx.doi.org/10.1093/pcp/pcm096DOI Listing
September 2007

Near-infrared spectroscopic method for real-time monitoring of pharmaceutical powders during voiding.

Appl Spectrosc 2007 May;61(5):490-6

Department of Chemistry, University of Puerto Rico-Mayagüez Campus, Mayagüez, Puerto Rico.

A near-infrared (NIR) spectroscopic method has been developed to monitor flowing pharmaceutical powders during their voiding and detect post-blending segregation. The method is capable of providing both chemical and physical information (particle size differences) on the flowing pharmaceutical powders. Particle size differences are widely recognized as the predominant driver for segregation. Pharmaceutical powders may segregate following blending as they are voided down pipes to compressing machines, increasing the variability of the drug content and dissolution of the final product tablets because of segregation. NIR diffuse reflectance spectra of pharmaceutical powders were obtained following voiding through a six-foot pipe. Spectral subtraction was used to eliminate baseline differences but maintain particle size differences. The NIR spectra indicated differences in the particle size of the flowing powder. Particle size differences were also tracked throughout the voiding of pharmaceutical powders by plotting the absorbance at 1536 nm. The method was also applied to the voiding of two layers of lactose particles with different particle sizes. The system described in this report provides an approach to study post-blending segregation in pharmaceutical powders and other relevant materials.
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http://dx.doi.org/10.1366/000370207780807713DOI Listing
May 2007

Utilization and transport of mannitol in Olea europaea and implications for salt stress tolerance.

Plant Cell Physiol 2007 Jan 21;48(1):42-53. Epub 2006 Nov 21.

Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

Mannitol is one of the primary photosynthetic products and the major phloem-translocated carbohydrate in Olea europaea L., an important crop in the Mediterranean basin. Uptake of mannitol in heterotrophic cell suspensions of O. europaea was shown to be mediated by a 1 : 1 polyol : H+ symport system with a Km of 1.3 mM mannitol and a Vmax of 1.3 nmol min(-1) mg(-1) DW. Dulcitol, sorbitol and xylitol competed for mannitol uptake, whereas glucose and sucrose did not. Reverse transcription-PCR (RT-PCR) performed on mRNA extracted from cultured cells exhibiting high mannitol transport activity allowed the cloning of a partial O. europaea mannitol carrier OeMaT1. The Vmax of mannitol uptake and the amount of OeMaT1 transcripts increased along with polyol depletion from the medium, suggesting that the mannitol transport system may be regulated by its own substrate. Addition of 100-500 mM NaCl to cultured cells enhanced the capacity of the polyol : H+ symport system and the amount of OeMaT1 transcripts, whereas it strongly repressed mannitol dehydrogenase activity. Measurements of cell viability showed that mannitol-grown cells remained viable 24 h after a 250 and 500 mM NaCl pulse, whereas extensive loss of cell viability was observed in sucrose-grown cells. OeMaT1 transcripts increased throughout maturation of olive fruits, suggesting that an OeMaT is involved in the accumulation of mannitol during ripening of olive. Thus, mannitol transport and compartmentation by OeMaT are important to allocate this source of carbon and energy, as well as for salt tolerance and olive ripening.
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http://dx.doi.org/10.1093/pcp/pcl035DOI Listing
January 2007

Pathways of glucose regulation of monosaccharide transport in grape cells.

Plant Physiol 2006 Aug 9;141(4):1563-77. Epub 2006 Jun 9.

Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

Grape (Vitis vinifera) heterotrophic suspension-cultured cells were used as a model system to study glucose (Glc) transport and its regulation. Cells transported D-[14C]Glc according to simple Michaelis-Menten kinetics superimposed on first-order kinetics. The saturating component is a high-affinity, broad-specificity H+ -dependent transport system (Km = 0.05 mm). Glc concentration in the medium tightly regulated the transcription of VvHT1 (Vitis vinifera hexose transporter 1), a monosaccharide transporter previously characterized in grape berry, as well as VvHT1 protein amount and monosaccharide transport activity. All the remaining putative monosaccharide transporters identified so far in grape were poorly expressed and responded weakly to Glc. VvHT1 transcription was strongly repressed by Glc and 2-deoxy-D-Glc, but not by 3-O-methyl-D-Glc or Glc plus mannoheptulose, indicating the involvement of a hexokinase-dependent repression. 3-O-Methyl-D-Glc, which cannot be phosphorylated, and Glc plus mannoheptulose induced a decrease of transport activity caused by the reduction of VvHT1 protein in the plasma membrane without affecting VvHT1 transcript levels. This demonstrates hexokinase-independent posttranscriptional regulation. High Glc down-regulated VvHT1 transcription and Glc uptake, whereas low Glc increased those parameters. Present data provide an example showing control of plant sugar transporters by their own substrate both at transcriptional and posttranscriptional levels. VvHT1 protein has an important role in the massive import of monosaccharides into mesocarp cells of young grape berries because it was localized in plasma membranes of the early developing fruit. Protein amount decreased abruptly throughout fruit development as sugar content increases, consistent with the regulating role of Glc on VvHT1 expression found in suspension-cultured cells.
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http://dx.doi.org/10.1104/pp.106.080804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1533936PMC
August 2006
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