Membrane water permeability and aquaporin expression increase during growth of maize suspension cultured cells

Aquaporins (AQPs) are water channels that allow cells to rapidly alter their membrane water permeability. A convenient model for studying AQP expression and activity regulation is Black Mexican Sweet (BMS) maize cultured cells. In an attempt to correlate membrane osmotic water permeability coefficient (P_f) with AQP gene expression, we first examined the expression pattern of 33 AQP genes using macro-array hybridization. We detected the expression of 18 different isoforms representing the four AQP subfamilies, i.e. eight plasma membrane (PIP), five tonoplast (TIP), three small basic (SIP) and two NOD26-like (NIP) AQPs. While the expression of most of these genes was constant throughout all growth phases, mRNA levels of ZmPIP1;3 , ZmPIP2;1 , ZmPIP2;2, ZmPIP2;4 and ZmPIP2;6 increased significantly during the logarithmic growth phase and the beginning of the stationary phase. The use of specific anti-ZmPIP antisera showed that the protein expression pattern correlated well with mRNA levels. Cell pressure probe and protoplast swelling measurements were then performed to determine the P_f. Interestingly, we found that the P_f were significantly increased at the end of the logarithmic growth phase and during the steady-state phase compared to the lag phase, demonstrating a positive correlation between AQP abundance in the plasma membrane and the cell P_f.     

Blood plasma model predictions for the proposed bone-seeking radiopharmaceutical [Sn]Sn(IV)-N,N′,N′-trimethylenephosphonate-poly(ethyleneimine)

In an attempt to elucidate the in vivo stability of the prospective radiopharmaceutical [^117mSn]Sn(IV)-PEI-MP, where PEI-MP stands for N,N′,N′-trimethylenephosphonate-polyethyleneimine, glass electrode potentiometry was used to determine the stability constants of the Sn⁴⁺ ion as complexed with a variety of physiological amino acids. In addition, linear free energy relationship (LFER) correlation plots were used to extrapolate the constants of the major blood plasma ligands, based on data from Cu²⁺, Pb²⁺, and Zn²⁺. In so doing, a thermodynamic model of blood plasma was established for Sn⁴⁺ from which the complexation tendencies of Sn⁴⁺ were predicted in the event of the intravenous administration of such a drug. It was found that the Sn(IV)-PEI-MP could succumb to competition by the glutamine amino acid, which forms more stable complex(es), whilst the PEI-MP gets taken up largely by Ca²⁺. Also, this study shows the value of the in vitro experiments and modeling performed for radiopharmaceutical research and for attempts to reduce the number of animal experiments.     

Morphofunctional study of 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA)‐induced differentiation of U937 cells under exposure to a 6 mT static magnetic field

This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F‐actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca²⁺]_i was evaluated with a spectrophotometer. The degree of differentiation in SMF‐exposed cells was lower than that of non‐exposed cells, the difference being exposure time‐dependent. SMF‐exposed cells showed cell shape and F‐actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non‐exposed, TPA‐stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca²⁺]_i could be one of the causes of the above‐described changes. Bioelectromagnetics 30:352–364, 2009. © 2009 Wiley‐Liss, Inc.     

Biochemical investigation of the effects of human platelet releasates on human articular chondrocytes

The aim of the present study was to demonstrate the mitogenic and differentiating properties of platelet‐rich plasma releasates (PRPr) on human chondrocytes in mono‐ and three‐dimensional cultures. In order to assess if PRPr supplementation could maintain the chondrocyte phenotype or at least inhibit the cell de‐differentiation even after several days in culture, we performed a proteomic study on several cell cultures independently grown, for different periods of time, in culture medium with FCS, human serum (HS), and releasates obtained from PRP and platelet‐poor plasma (PPP). We found that PRP treatment actually induced in chondrocytes the expression of proteins (some of which novel) involved in differentiation. J. Cell. Biochem. 108: 1153–1165, 2009. © 2009 Wiley‐Liss, Inc.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Estimation of antioxidant capacity against pathophysiologically relevant oxidants using Pyrogallol Red

Peroxynitrite and hypochlorite are oxidants relevant in many pathological situations. We propose a simple spectrophotometric assay to determine antioxidant capacity against hypochlorite and peroxynitrite based on protection against Pyrogallol Red decolorization. The assay can be performed on a microplate and requires minute amounts of material. Standard antioxidants show different reactivities for both oxidants. Antioxidant capacity of blood plasma (anticoagulated with EDTA) of healthy persons was found to be 559 ± 49 μmol/l and 11.6 ± 1.2 mmol/l of ascorbic acid equivalents for peroxynitrite and hypochlorite, respectively.     

Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n = 3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean ± SEM; 0.51 ± 0.21, 0.35 ± 0.09, p < 0.05), (2) lower (p < 0.05) secretion of IGF-binding protein 4 (9.6 ± 1.2 vs. 17.4 ± 2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6 ± 1.9 vs. 39.1 ± 6.3 μIU/ml), (3) lower protein expression of IGF2 “clearance” receptor in cell lysate (0.28 ± 0.03 vs. 0.53 ± 0.02 OD U/mm², p < 0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.     

The effect of packing hydrophilization on bacterial attachment and the relationship with the performance of biotrickling filters

Many bioprocesses depend on the effective formation of a biofilm on a solid support. In the present study, three different surface treatments (sandblasting, pure‐O₂ plasma, and He–O₂ plasma treatments) were conducted on polypropylene (PP) Pall rings used as a support in biotrickling filters for air pollution control. The intent was to modify the ring surface and/or electrochemical properties in order to possibly improve cell adhesion, wetting properties, and possibly reduce the start‐up time and increase the performance of the biotrickling filters. The surface treatments were found to generally increase the hydrophilicity and the zeta potential of the surfaces. However, the startup and performance of lab‐scale biotrickling filters packed with treated Pall rings were not significantly different than the control with untreated rings. Cell and colloid deposition experiments conducted in flow cells showed that the treated surfaces and the hydrodynamic conditions were not favorable for cell deposition indicating that there could be significant opportunities for improving packings used in environmental bioprocess applications. Biotechnol. Bioeng. 2009;103: 1060–1067. © 2009 Wiley Periodicals, Inc.     

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca²⁺ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C^α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.