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The role of sulfhydryl groups in alanine transport by lyophilized brush border membrane vesiclesStevens, Bruce Russell. Preston, Robert Leslie. January 1980 (has links)
Thesis (Ph. D.)--Illinois State University, 1980. / Title from title page screen, viewed Mar. 4, 2005. Dissertation Committee: Robert Preston (chair), John Frehn, Harry Huizinga, Arlan Richardson, Jim Tone. Includes bibliographical references (leaves 208-222) and abstract. Also available in print.
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Structure, membrane association, and processing of meprin subunits /Marchand, Petra, January 1994 (has links)
Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 147-156). Also available via the Internet.
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Exposure to Engineered Nanomaterial Results in Disruption of Brush Borders in Epithelia Models in vitroJanuary 2014 (has links)
abstract: Engineered nanoparticles (NP; 10-9 m) have found use in a variety of consumer goods and medical devices because of the unique changes in material properties that occur when synthesized on the nanoscale. Although many definitions for nanoparticle exist, from the perspective of size, nanoparticle is defined as particles with diameters less than 100 nm in any external dimension. Examples of their use include titanium dioxide added as a pigment in products intended to be ingested by humans, silicon dioxide NPs are used in foods as an anticaking agent, and gold or iron oxide NPs can be used as vectors for drug delivery or contrast agents for specialized medical imaging. Although the intended use of these NPs is often to improve human health, it has come to the attention of investigators that NPs can have unintended or even detrimental effects on the organism. This work describes one such unintended effect of NP exposure from the perspective of exposure via the oral route. First, this Dissertation will explain an event referred to as brush border disruption that occurred after nanoparticles interacted with an in vitro model of the human intestinal epithelium. Second, this Dissertation will identify and characterize several consumer goods that were shown to contain titanium dioxide that are intended to be ingested. Third, this Dissertation shows that sedimentation due to gravity does not artifactually result in disruption of brush borders as a consequence of exposure to food grade titanium dioxide in vitro. Finally, this Dissertation will demonstrate that iron oxide nanoparticles elicited similar effects after exposure to an in vitro brush border expressing model of the human placenta. Together, these data suggest that brush border disruption is not an artifact of the material/cell culture model, but instead represents a bona fide biological response as a result of exposure to nanomaterial. / Dissertation/Thesis / Doctoral Dissertation Biology 2014
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Characterization of methionine transport in bovine intestinal brush border membrane vesiclesDahl, Geoffrey Eliot January 1987 (has links)
Characteristics of methionine uptake by brush border membrane vesicles of bovine small intestine were investigated. Alkaline phosphatase marked the brush border membrane fraction obtained through differential centrifugation followed by a sucrose gradient. This preparation yielded a 10-fold enrichment of activity over homogenate. Methionine uptake was found to be into an osmotically active space. A binding constant of 75.4 pmol/mg membrane protein was determined. A significant (p<.05) sodium stimulation of methionine uptake was observed. This indicated active (energy-dependent) transport in addition to the diffusive component of intravesicular mention accumulation. Decreasing the pH of buffer medium significantly (p<.05) depresses methionine transport. A K<sub>m</sub> = .114 mM and V<sub>MAX</sub> = 56.5 pmol/s/mg membrane protein were ascertained for methionine. / M.S.
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Structure, membrane association, and processing of meprin subunitsMarchand, Petra 06 June 2008 (has links)
Meprins are oligomeric cell-surface metalloproteinases that are expressed at high concentrations in the renal brush border membranes of mice. Meprins consist of two types of subunits, α and β, which are the products of two different genes. The β subunit cDNA was cloned and sequenced from mouse kidneys, and the membrane association and the in vivo proteolytic processing of mouse meprin subunits were investigated. The primary translation product of the meprin β subunit is composed of 704 amino acids, and contains several domains, including a signal sequence at the NH₂-terminus, a prosequence, a protease domain, an adhesion domain (MAM domain), an epidermal growth factor-like domain, a potential transmembrane-spanning domain, and a short cytoplasmic tail. The B subunit is evolutionarily related to the α subunit. The α and β subunit share about 42% overall sequence identity, and have a similar arrangement of functional domains; however, a 56 amino acid segment near the COOH-terminus of a is missing in β, and the signal sequences, transmembrane and cytoplasmic domains share no significant sequence similarity. The protease domains of o and B are 55% identical. NH₂-terminal protein sequencing of detergent-solubilized meprin subunits from mouse kidneys showed that the prosequence in a is removed in the mature subunit. By contrast, only the signal sequence is removed from the mature β subunit NH₂-terminus, and the β subunit retains the prosequence. Further, the mature α subunit, but not the β subunit, is proteolytically processed at the COOH-terminus and does not contain the transmembrane and the EGF-like domains encoded by the meprin α cDNA. The β subunit is a type I integral membrane protein. By contrast, α does not transverse the membrane, and its membrane association depends on disulfide bonds. The oligomeric organization of membrane-bound meprins was analyzed by SDSPAGE under non-reducing conditions, and by isoelectric focusing. ICR mouse kidneys express αβ heterodimers and α₂ homodimers; C3H/He mice contain β₂ dimers. Transfection of COS-1 cells with the full-length meprin α subunit cDNA resulted in the secretion of meprin dimers into the culture medium, indicating that the COOH-terminal transmembrane domain of meprin α subunits is posttranslationally removed from the protein in COS-1 cells, as it is in mouse kidney cells. Replacement of the COOH-terminal 137 amino acids of a with the COOH-terminus of B, or deletion of the 56 amino acid inserted domain in α, resulted in mutant proteins that were not secreted into the medium, but rather were membrane-bound, indicating that the inserted domain of α is essential for proteolytic cleavage and secretion. Deletion of the COOH-terminal 133 residues of a did not affect meprin α dimerization or intracellular transport. The meprin a subunits secreted from transfected COS-1 cells were catalytically inactive, but could be activated by limited proteolysis with trypsin. Thus, processing at the NH,-terminus differed in COS-1 cells and in mouse kidney. COS-1 cells did not remove the prosequence from the α subunit protein, and removal of the prosequence was essential for catalytic activity of the a subunit. These results have implications for the biosynthesis and regulation of a cell surface proteinase, and thus relate to the elucidation of the mechanisms by which biological events at the cell surface are regulated. / Ph. D.
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Imagerie par microscopie à force atomique de toxines Cry1 de Bacillus thuringiensis interagissant avec des membranes apicales de l'intestin de Manduca sextaLaflamme, Eric January 2008 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.
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Imagerie par microscopie à force atomique de toxines Cry1 de Bacillus thuringiensis interagissant avec des membranes apicales de l'intestin de Manduca sextaLaflamme, Eric January 2008 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal
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Caractérisation du co-transporteur Na+/myo-inositol SMIT2 dans les membranes en bordure en brosse de rein de lapin et d’intestin de ratAouameur, Rym 03 1900 (has links)
Le myo-inositol (MI) est un soluté organique impliqué dans diverses
fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également
un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire
responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au
transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de
protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI
par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de
lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus
laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a
appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2
est principalement localisé dans le cortex. Ces résultats ont été confirmés par
immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition
sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous
avons démontré que SMIT2 semble le seul responsable du transport luminal de MI
dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de
l’intestin, des études de transport de MI radioactif ont démontré une absence de
transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI
très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin
de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le
rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des
entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de
l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur
SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus
laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin
et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le
MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est
ii
également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la
constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus
petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également
testé la capacité de certains transporteurs de sucre présents à la surface des
membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces
transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif
transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport.
Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas
effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est
incapable de transporter le MI. / Myo-inositol (MI) is an organic solute involved in various aspects of cell
physiology, including cell signaling. It is also known as a compatible osmolyte.
Three secondary active MI cotransporters have been identified; two are Na+-
coupled (SMIT1 and SMIT2) and one is H+-coupled (HMIT). The main aim of this
study was to characterize MI uptake throught SMIT2 as expressed in epithelial
cells and in Xenopus laevis oocytes. In order to achieve the characterization of this
transport system, we used purified brush border membrane vesicles (BBMv)
isolated from rabbit kidney and rat intestine. We first performed a quantification of
mRNA levels in rabbit kidney using real time PCR for both SMIT1 and SMIT2.
We found that SMIT1 is mainly expressed in the renal medulla while SMIT2 is
mainly localized in the renal cortex. This result was confirmed on Western blots
using an antibody raised against SMIT2. Through inhibition studies using selective
substrates for SMIT1 (inhibited by L-fucose) and SMIT2 (inhibited by D-chiroinositol),
we showed that SMIT2 seems to be responsible for all the apical
transport of MI into the proximal convoluted tubule with a Km of 57 ± 14 µM. By
transport studies we established that rabbit intestine seems to lack apical transport
of MI while rat intestine has a very active uptake of this molecule. qRT-PCR
quantification confirmed the absence of MI transporters in rabbit intestine. As for
kidney, SMIT2 seems to be the only transporter responsible for apical MI uptake
in enterocytes with a Km of 150 ± 40 µM. Functional analysis of rat SMIT2
activity, via electrophysiological studies in Xenopus oocytes, demonstrated
similarities to the activities of SMIT2 from rat intestine and rabbit kidney. SMIT2
displays high affinities for MI (270 ± 19 µM), DCI (310 ± 60 µM) and no affinity
for L-fucose. SMIT2 is very sensitive to phlorizin (Pz; 16 ± 7 µM). Although these
functional characteristics essentially confirmed those found in rat intestine, a
iv
discrepancy exists between the two systems studied. Indeed, the affinity constant
for glucose was approximately 40-fold lower in vesicles than in oocytes. We also
tested the ability of SGLT1 and GLUT5, other sugar transport systems present in
enterocytes apical membranes, to perform MI uptake. Because the inhibition of
these transporters did not alter radiolabeled MI uptake, we concluded that they had
no significant contribution to MI transport in rat intestine. Finally, the basolateral
efflux of MI was not mediated by GLUT2 because when expressed in oocytes, this
transporter was not able to transport MI.
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Viabilização do uso de Bt para o manejo do HLB dos citros via redução da população de Diaphorina citri / Feasibility of the Bt use for reduction of Diaphorina citri population and improved citrus HLB managementCunha, Tatiane da 03 April 2018 (has links)
A citricultura se destaca como uma das mais importantes atividades do agronegócio brasileiro, sendo o estado de São Paulo o principal produtor mundial de laranja e o maior exportador de suco concentrado e congelado. O psilídeo dos citros (Diaphorina citri) tornou-se nos últimos anos um dos mais importantes vetores na cultura, devido à transmissão de \'Candidatus Liberibacter asiaticus\' e \'Ca. L. americanus\', bactérias associadas ao huanglongbing (HLB), principal doença da citricultura atual. Uma vez que não há variedades comerciais de citros resistentes ao HLB, seu manejo é baseado no uso de mudas sadias, erradicação de plantas infectadas e pelo controle químico do vetor. No entanto, o custo elevado e os significativos danos ambientais causados pelos inseticidas químicos, associados à possibilidade da seleção de populações de psilídeos resistentes a esses produtos, têm levado à busca por estratégias alternativas de manejo do vetor do HLB que sejam mais adequadas e sustentáveis. Nosso grupo tem tentado contribuir nesse sentido, com a prospecção e caracterização de estirpes de Bacillus thuringiensis (Bt) patogênicos ao psilídeo. Sendo assim, os objetivos deste estudo foram: (a) Confirmar a capacidade endofítica e a patogenicidade de estirpes de Bt em seedlings e mudas de citros com diferentes combinações de copa/porta-enxerto; (b) Determinar a concentração letal (CL50) necessária para ocasionar mortalidade em populações de D. citri; (c) Estudar a interação entre toxinas Cry e receptores de membrana do intestino de ninfas e (d) Avaliar a compatibilidade das estirpes selecionadas com agrotóxicos comumente utilizados na citricultura. Os bioensaios realizados com seedlings e mudas de citros demonstraram que as estirpes de Bt (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 e S1989) translocaram endofiticamente nas plantas, mantendo sua viabilidade e, em sua maioria, o potencial patogênico para ninfas de D. citri. Para seedlings, os valores de mortalidade passaram de 80% para as estirpes S1302 e S1450. Foram observados esporos e células vegetativas de B. thuringiensis subsp. kurstaki expressando green fluorescent protein (Btk::GFP), visualizados por microscopia de fluorescência, aderidos principalmente aos elementos de vasos e no xilema, obtidas de amostras seccionadas transversal e longitudinalmente, de caule e raiz de seedlings e mudas de laranjeira Pera. A CL50 e CL80 para a estirpe S1302 foi de 4,92 × 104 esporos/mL e 6,63 × 107 esporos/mL, respectivamente. Já para a estirpe S1450, 50% de mortalidade das ninfas testadas foi estimada em 2,19 × 104 esporos/mL, e a CL80 foi de 6,18 × 108 esporos/mL, quando utilizadas suspensões de esporos da bactéria diretamente no substrato de seedlings de laranjeira Pera. Os ensaios de ligação demonstraram que todas as toxinas Cry presentes nas estirpes S1302, S1450 e S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B e Cry11) interagiram com os receptores de membrana intestinal, brush border membrane vesicles (BBMV\'s), obtidos de ninfas de D. citri. Ensaios in vivo evidenciaram que as estirpes S1302 e S1450 mostraram-se compatíveis com todos os agrotóxicos comumente utilizados na citricultura, ainda que os produtos à base de cobre e o inseticida Dimetoato tenham sido deletérios em aplicações diretas na bactéria in vitro. Esses dados evidenciam a possibildade do uso de Bt como bioinseticida no manejo integrado do HLB. / Citriculture is one the most important activities of Brazilian agribusiness, and the State of São Paulo being the world\'s leading orange producer and the largest juice exporter. Asian citrus psyllid - ACP (Diaphorina citri) has become one of the most important vectors in the citriculture in recent years, due to the transmission of \'Candidatus Liberibacter asiaticus\' and \'Ca. L. americanus\', bacteria associated with huanglongbing (HLB), the main citrus disease worldwide. Nowadays there are no commercial citrus varieties resistant to HLB, and its management is based on the use of healthy nursery citrus trees, eradication of symptomatic planst from the orchards, and vector chemical control. However, excessive cost and environmental damage due to application of chemical insecticides associated with the possibility of selection of ACP resistant populations, have led researchers to persue alternative strategies for the management of HLB. Our group has contributed with the screening of Bacillus thuringiensis (Bt) strains with potential against ACP. Therefore, our objectives were: (a) to confirm Bt endophytic translocation in citrus seedlings and nursery trees with different scion-rootstock combinations and to evaluate their pathogenicity against D. citri; (b) to estimate the lethal concentration (LC50 and LC80) to D. citri nymphs; (c) to study the interaction between Cry toxins and brush border membrane vesicle (BBMV) of the midgut D. citri nymphs, and (d) to evaluate the compatibility of pesticides with the selected Bt strains. The bioassays with citrus seedlings and nursery trees demonstrated that the Bt strains (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 and S1989) translocated from roots to young leaves, maintaining their viability and pathogenicity against D. citri nymphs. For the seedlings, we found mortality values up to 80% for strains S1302 and S1450. Btk::GFP spores and vegetative cells were visualized by fluorescence microscopy in citrus seedlings and nursery trees adhered mainly to vessels and xylem, from roots to stems, in cross-section analyses. LC50 and LC80 for strain S1302 were 4.92 × 104 spores/mL and 6.63 × 107 spores/mL, respectively. For strain S1450, 50% mortality of nymphs tested was estimated at 2.19 × 104 spores/mL, and LC80 was 6.18 × 108 spores/mL, when bacterial spore suspensions were applied to citrus seedlings. Binding assays demonstrated that all Cry toxins present in strains S1302, S1450 and S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B and Cry11) interacted with the BBMV obtained from D. citri nymphs. In vivo assays showed that strains S1302 and S1450 were compatible with all pesticides commonly used in citrus orchards, althoug copper-based pesticides and dimethoate insecticide were incompatible in vitro with Bt strains. Our results demonstrate the potential of using Bt as systemic bioinsecticide in the future in HLB management.
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Caractérisation du co-transporteur Na+/myo-inositol SMIT2 dans les membranes en bordure en brosse de rein de lapin et d’intestin de ratAouameur, Rym 03 1900 (has links)
Le myo-inositol (MI) est un soluté organique impliqué dans diverses
fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également
un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire
responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au
transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de
protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI
par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de
lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus
laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a
appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2
est principalement localisé dans le cortex. Ces résultats ont été confirmés par
immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition
sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous
avons démontré que SMIT2 semble le seul responsable du transport luminal de MI
dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de
l’intestin, des études de transport de MI radioactif ont démontré une absence de
transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI
très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin
de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le
rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des
entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de
l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur
SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus
laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin
et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le
MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est
ii
également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la
constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus
petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également
testé la capacité de certains transporteurs de sucre présents à la surface des
membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces
transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif
transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport.
Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas
effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est
incapable de transporter le MI. / Myo-inositol (MI) is an organic solute involved in various aspects of cell
physiology, including cell signaling. It is also known as a compatible osmolyte.
Three secondary active MI cotransporters have been identified; two are Na+-
coupled (SMIT1 and SMIT2) and one is H+-coupled (HMIT). The main aim of this
study was to characterize MI uptake throught SMIT2 as expressed in epithelial
cells and in Xenopus laevis oocytes. In order to achieve the characterization of this
transport system, we used purified brush border membrane vesicles (BBMv)
isolated from rabbit kidney and rat intestine. We first performed a quantification of
mRNA levels in rabbit kidney using real time PCR for both SMIT1 and SMIT2.
We found that SMIT1 is mainly expressed in the renal medulla while SMIT2 is
mainly localized in the renal cortex. This result was confirmed on Western blots
using an antibody raised against SMIT2. Through inhibition studies using selective
substrates for SMIT1 (inhibited by L-fucose) and SMIT2 (inhibited by D-chiroinositol),
we showed that SMIT2 seems to be responsible for all the apical
transport of MI into the proximal convoluted tubule with a Km of 57 ± 14 µM. By
transport studies we established that rabbit intestine seems to lack apical transport
of MI while rat intestine has a very active uptake of this molecule. qRT-PCR
quantification confirmed the absence of MI transporters in rabbit intestine. As for
kidney, SMIT2 seems to be the only transporter responsible for apical MI uptake
in enterocytes with a Km of 150 ± 40 µM. Functional analysis of rat SMIT2
activity, via electrophysiological studies in Xenopus oocytes, demonstrated
similarities to the activities of SMIT2 from rat intestine and rabbit kidney. SMIT2
displays high affinities for MI (270 ± 19 µM), DCI (310 ± 60 µM) and no affinity
for L-fucose. SMIT2 is very sensitive to phlorizin (Pz; 16 ± 7 µM). Although these
functional characteristics essentially confirmed those found in rat intestine, a
iv
discrepancy exists between the two systems studied. Indeed, the affinity constant
for glucose was approximately 40-fold lower in vesicles than in oocytes. We also
tested the ability of SGLT1 and GLUT5, other sugar transport systems present in
enterocytes apical membranes, to perform MI uptake. Because the inhibition of
these transporters did not alter radiolabeled MI uptake, we concluded that they had
no significant contribution to MI transport in rat intestine. Finally, the basolateral
efflux of MI was not mediated by GLUT2 because when expressed in oocytes, this
transporter was not able to transport MI.
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