Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2; 1) by cytosolic pH

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His131 residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pHin dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pHinfo:eu-repo/semantics/publishedVersio

Conjugated linoleic acid reduces permeability and fluidity of adipose plasma membranes from obese Zucker rats

NOTICE: this is the author’s version of a work that was accepted for publication in Biochemical and Biophysical Research Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochemical and Biophysical Research Communications. July 2010; 398 (2): 199-204.Conjugated linoleic acid (CLA) is a dietary fatty acid frequently used as a body fat reducing agent whose effects upon cell membranes and cellular function remain unknown. Obese Zucker rats were fed atherogenic diets containing saturated fats of vegetable or animal origin with or without 1% CLA, as a mixture of cis(c)9,trans(t)11 and t10,c12 isomers. Plasma membrane vesicles obtained from visceral adi- pose tissue were used to assess the effectiveness of dietary fat and CLA membrane incorporation and its outcome on fluidity and permeability to water and glycerol. A significant decrease in adipose membrane fluidity was correlated with the changes observed in permeability, which seem to be caused by the incor- poration of the t10,c12 CLA isomer into membrane phospholipids. These results indicate that CLA supple- mentation in obese Zucker rats fed saturated and cholesterol rich diets reduces the fluidity and permeability of adipose membranes, therefore not supporting CLA as a body fat reducing agent through membrane fluidification in obese fat consumers

Biophysical assessment of human aquaporin-7 as a water and glycerol channel in 3T3-L1 adipocytes

The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes

Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation

NOTICE: this is the author’s version of a work that was accepted for publication in Biochemical and Biophysical Research Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochemical and Biophysical Research Communications. May 2011; 408 (3): 477-481.In general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (Pf) at 23 ºC was (2.89 ± 0.37) × 10-2 and (5.12 ± 0.61) × 10-2 cm s-1 for human and bovine cells respectively, with similar activation energies for water transport. Glycerol permeability (Pgly) for human ((1.37 ± 0.26) × 10-5 cm s-1) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) ×10-8 cm s-1) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger Pf found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high Ea for transport observed in ruminants

Membrane tension regulates water transport in yeast

Evidence that membrane surface tension regulates water fluxes in intact cells of a Saccharomyces cerevisiae strain overexpressing aquaporin AQY1 was obtained by assessing the osmotic water transport parameters in cells equilibrated in different osmolarities. The osmotic water permeability coefficients (Pf) obtained for yeast cells overexpressing AQY1 incubated in low osmolarity buffers were similar to those obtained for a double mutant aqy1aqy2 and approximately three times lower (with higher activation energy, Ea) than values obtained for cells incubated in higher osmolarities (with lower Ea). Moreover, the initial inner volumes attained a maximum value for cells equilibrated in lower osmolarities (below 0.75 M) suggesting a preswollen state with the membrane under tension, independent of aquaporin expression. In this situation, the impairment of water channel activity suggested by lower Pf and higher Ea could probably be the first available volume regulatory tool that, in cooperation with other osmosensitive solute transporters, aims to maintain cell volume. The results presented point to the regulation of yeast water channels by membrane tension, as previously described in other cell system

Effect of dietary conjugated linoleic acid isomers on water and glycerol permeability of kidney membranes

NOTICE: this is the author’s version of a work that was accepted for publication in Biochemical and Biophysical Research Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochemical and Biophysical Research Communications. May 2009; 383(1): 108-112.Conjugated linoleic acid (CLA) refers to a group of positional and geometrical isomers of linoleic acid in which the double bonds are conjugated. Dietary CLA has been associated with various health benefits although details of its molecular mode of action remain elusive. The effect of CLA supplemented to palm oil-based diets in Wistar rats, as a mixture of both or isolated c9,t11 and t10,c12 isomers, was examined on water and glycerol membrane permeability of kidney proximal tubule. Although water permeability was unaltered, an increase in glycerol permeability was obtained for the group supplemented with CLA mixture, even though the activation energy for glycerol permeation remained high. This effect was correlated with an increased CLA isomeric membrane incorporation for the same dietary group. These results suggest that diet supplementation with CLA mixture, in contrast to its individual isomers, may enhance membrane fluidity subsequently raising kidney glycerol reabsorption

On the evaluation of strain energy release rate of cement-bone bonded joints under mode II loading

Bone cements based on poly(methylmethacrylate) (PMMA) are primarily used in joint replacement surgeries. In the fixation of joint replacement, the self-curing cement fills constitutes a very important interface. To understand and improve the interaction between cortical bone and bone cement it is essential to characterize the mechanical properties of cement-bone bonded joints in full detail. In this study, the end-notched flexure test was used in the context of pure mode II fracture characterisation of cement-bone bonded joints. A data reduction scheme based on crack equivalent concept was employed to overcome the difficulties inherent to crack length monitoring during damage propagation. A finite element method combined with a cohesive zone model was first used to validate numerically the adopted method. The procedure was subsequently applied to experimental results to determine the fracture toughness of cement-bone bonded joints under pure mode II loading. The consistency of the obtained results leads to the conclusion that the adopted procedure is adequate to carry out fracture characterisation of these joints under pure mode II loading. The innovative aspect of the present work lies in the application of cohesive zone modelling approach to PMMA-based cement-bone bonded joints

Fracture characterisation of bone-cement bonded joints under mode I loading

Over the years, many techniques have been developed for the stabilisation of bone fractures. The study of the adhesion of bone-to-bone cement is an important step towards the development of new immobilization systems. Although bone cement has been used for more than fifty years, very few studies have been performed regarding the evaluation of fracture properties. In this work, numerical and experimental investigations were conducted to evaluate the strain energy release rate under mode I loading in a bone-cement bonded joint, using the Double Cantilever Beam (DCB) test. Cohesive zone laws were also measured combining the finite element method with non-linear elastic fracture mechanics. This has been made in a cortical bone bonded joint with poly- methylmethacrylate (PMMA). Consistent results have been obtained regarding fracture toughness in a widely used bone-to-bone cement joint in many biomedical applications.The first author acknowledges the Portuguese (FCT) for the conceded financial support through the reference grant PTDC/EME-SIS/28225/2017. M.F.S.M. de Moura acknowledges the ‘Laboratório Associado de Energia, Transportes e Aeronáutica’ (LAETA) for the financial support by the project UID/EMS/50022/2020. The corresponding author acknowledges FCT for the conceded financial support through the reference projects PTDC/EME-SIS/28225/2017 and UID/EEA/04436/2019.Funding: Portuguese Foundation for Science and Technology for MsC grant of the first author, and research project PTDC/EME-SIS/28225/2017

Osteosynthesis metal plate system for bone fixation using bicortical screws: numerical–experimental characterization

This study reports the numerical and experimental characterization of a standard immobilization system currently being used to treat simple oblique bone fractures of femoral diaphyses. The procedure focuses on the assessment of the mechanical behavior of a bone stabilized with a dynamic compression plate (DCP) in a neutralization function, associated to a lag screw, fastened with surgical screws. The non-linear behavior of cortical bone tissue was revealed through four-point bending tests, from which damage initiation and propagation occurred. Since screw loosening was visible during the loading process, damage parameters were measured experimentally in independent pull-out tests. A realistic numerical model of the DCP-femur setup was constructed, combining the evaluated damage parameters and contact pairs. A mixed-mode (I+II) trapezoidal damage law was employed to mimic the mechanical behavior of both the screw–bone interface and bone fractures. The numerical model replicated the global behavior observed experimentally, which was visible by the initial stiffness and the ability to preview the first loading peak, and bone crack satisfactorily.This research was funded by the Portuguese Foundation for Science and Technology (FCT), grant numbers SFRH/BD/143736/2019, UIDB/CVT/00772/2020, LA/P/0059/2020, UIDB/04033/2020, PTDC/EME-SIS/28225/2017, UID/EEA/04436/2019 and Laboratório Associado de Energia, Transportes e Aeronáutica (LAETA), grant number UID/EMS/50022/2020