Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture — primary culture cells markedly differ from fourth-passage cells

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase⁺/74% Thy-1/CD90⁺ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.     

Do detached root-cap cells influence bacteria associated with maize roots?

Abstract. A model rhizosphere has been used which consisted of detached root-cap cells of maize in their surrounding root-cap mucilage on the surface of Noble agar. These cells were co-cultured for periods up to 32 d with eight different bacterial isolates from soil-grown roots and surrounding soil and two laboratory cultures. Cap cells were unaffected by the bacteria. There were five different type-specific responses of the bacteria in proximity to the cap cells. There were, strong growth inhibition ( Rhizobium sp. and Escherichia coli ), strong stimulation ( Pseudomonas fluorescens , laboratory strain), mixed weak inhibition or stimulation ( Pseudomonas fluorescens , field isolate), early inhibition followed by strong stimulation then spore formation ( Bacillus spp.), no effect ( Streptomyces sp. and Cytophaga sp.). It is concluded that detached root-cap cells are actively involved in the establishment of characteristic rhizosphere bacterial microflora.     

Sucrose isomerase and its mutants from Erwinia rhapontici can synthesise α-arbutin

Sucrose isomerase (SI) from Erwinia rhapontici is an intramolecular isomerase that is normally used to synthesise isomaltulose from sucrose by a mechanism of intramolecular transglycosylation. In this study, it was found that SI could synthesise α-arbutin using hydroquinone and sucrose as substrates, via an intermolecular transglycosylation reaction. Five phenylalanine residues (F185, F186, F205, F297, and F321) in the catalytic pocket of SI were chosen for sitedirected mutagenesis. Mutants F185I, F321I, and F321W, whose hydrolytic activities were enhanced after the mutation, could synthesise α-arbutin through intermolecular transglycosylation with a more than two-fold increase in the molar transfer ratio compared with wild type SI. The F297A mutant showed a strong ability to synthesise a novel α-arbutin derivative and a four-fold increase in its specific activity for intermolecular transglycosylation over the wild type. Our findings may lead to a new way to synthesise novel glucoside products such as α-arbutin derivatives by simply manipulating the Phe residues in the catalytic pocket. From the structure superposition, our strategy of manipulating these Phe residues may be applicable to other similar transglycosylating enzymes.     

Inorganic mercury in mammary cells: viability,metal uptake but efflux?

The viability, cellular uptake and subcellular distribution of heavy metal Hg, were determined in human mammary cell lines (MCF-7, MDA-MB-231 and MCF-10A). It was observed that Hg had the capacity of being excluded from the cells with a different type of possible transporters. MCF-7 cells showed the lowest viability, while the other two cell lines were much more resistant to Hg treatments. The intracellular concentration of Hg was higher at lower exposure times in MCF-10A cells and MCF-7 cells; but as the time was increased only MDA-MB-231 showed the capacity to continue introducing the metal. In MCF-7 and MCF-10A cells the subcellular distribution of Hg was higher in cytosolic fraction than nucleus and membrane, but MDA-MB-231 showed membrane and nucleus fraction as the enriched one. The analysis of RNA-seq about the genes or family of genes that encode proteins which are related to cytotoxicity of Hg evidenced that MCF-10A cells and MCF-7 cells could have an active transport to efflux the metal. On the contrary, in MDA-MB-231 no genes that could encode active transporters have been found.     

Adult stem cells derived from skeletal muscle — biology and potential

Skeletal muscle contains at least two distinct populations of adult stem cells — satellite cells and multipotent muscle-derived stem cells. Monopotential satellite cells are located under the basal lamina of muscle fibers. They are capable of giving rise only to cells of myogenic lineage, which play an important role in the processes of muscle regeneration. Multipotent muscle-derived stem cells are considered to be predecessors of the satellite cells. Under proper conditions, both in vitro and in vivo, they undergo myogenic, cardiogenic, chondrogenic, osteogenic and adipogenic differentiation. The main purpose of the present article is to summarize current information about adult stem cells derived from skeletal muscle, and to discuss their isolation and in vitro expansion techniques, biological properties, as well as their potential for regenerative medicine.     

GM maize from site-specific recombination technology,what next?

The term plant genetic engineering has long conveyed a highly efficient and precise process for the manipulation of plant genomes. For nearly two decades, research on recombinase-based applications has steadily advanced the surgical capabilities of plant genome rearrangements. Once considered interesting laboratory exercises, a first crop plant derived from this type of DNA acrobatics is heading to market. Originally configured for a specific application, to remove a selectable marker, it could be the first of more to come - and not just market-free plants.     

Metalloproteinase-mediated Shedding of Integrin β2 promotes macrophage efflux from inflammatory sites

Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin β2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin β2 from mouse macrophages are demonstrated. Soluble integrin β2 is primarily released as a heterodimeric complex with αM that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin β2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin β2 is accelerated, and antibody disruption of integrin β2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin β2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.     

B cells biology in systemic lupus erythematosus—from bench to bedside

Systemic lupus erythematosus(SLE) is a debilitating autoimmune disease that can involve multi-organs. B cells play a central role in the immunopathogenesis via antibody-dependent and antibody-independent ways. Excessive autoantibodies production, hyperresponsiveness and prolonged survival of autoreactive B cells are characteristics of SLE. In this article, mechanisms of self-tolerance loss of B cells and promising B cell-targeting therapies in lupus are reviewed and discussed.     

iPS cells—Alternative pluripotent cells to embryo stem cells

<正>Since the first murine and human embryonic stem cell lines were established by Drs. Evans and Kaufman [1] and Thomson et al. [2], respectively, great progress has been make in the field of     

Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

Mechanism of passive malic-acid efflux from vacuoles of the CAM plantKalanchoë daigremontiana

Summary An analysis was carried out of the mechanism of malic-acid efflux from vacuoles of mesophyll cells of the crassulacean acid metabolism (CAM) plantKalanchoë daigremontiana. Following its accumulation in the vacuole as a result of nocturnal CO₂ fixation, the malic acid is passively transported back across the tonoplast in the subsequent light period and is decarboxylated in the cytoplasm. Malic-acid efflux was studied using leaf slices in solution or by following malic-acid utilization (deacidification) in leaves of intact plants. Samples of leaf-cell sap were taken at different times during the day-night rhythm to establish the relation between cell-sap pH and malate content. From the empirically determined pK values for malic acid in the cell sap, it was then possible to calculate the proportion of malate existing as the undissociated acid (H₂mal⁰) and in the anionic forms (Hmal^1– and mal^2–) for all times during the CAM rhythm. In leaf-slice experiments it has been found that the rate of malic-acid efflux increases exponentially with the malic-acid content of the tissue. This is shown to be related to the increasing amounts of H₂mal⁰ present at high malic-acid contents. At low malic-acid contents (<65 mol m^–3), when H₂mal⁰ is not present in significant amounts, efflux must be in the form of Hmal^–1 and/or mal^2–. At high malic-acid contents it is suggested that efflux occurs predominantly in the form of passive, noncatalyzed diffusion of H₂mal⁰ across the tonoplast by a lipid-solution mechanism. This is supported by the fact that the slope of the curve relating efflux to H₂mal⁰ concentration, when corrected for the presumed contributions from Hmal^1– and mal^2– transport and plotted on a log-log basis, approaches 1.0 at the highest malic-acid contents. Moreover, the permeability coefficient required to be consistent with such a mechanism is similar to that estimated from a Collander plot, using the partition coefficient of malic acid between ether and water. We suggest that may be important in determining the maximum amounts of malic acid that can be accumulated during the CAM rhythm.     

Cardiomyocyte-like cells differentiation from non β-catenin expression mesenchymal stem cells

Recent studies have shown that block wnt/β-catenin signaling pathway is integrant for cardiomyocytes differentiation from bone marrow mesenchymal stem cells (MSCs). By transducing the MSCs with lentivirus which contain β-catenin interference RNA, we screened out the non β-catenin expression clone. In the establishment of knockdown β-catenin in MSCs, we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (salB), and cardiomyocytes lysis medium (CLM) in inducing MSCs to differentiate into cardiomyocyte-like cells. A method for culturing MSCs and cardiomyocytes was established. Purified MSCs were investigated by flow cytometry. The MSCs were positive for CD90 and CD29, but negative for CD34 and CD45. Meanwhile, the cardiomyocytes contracted spontaneously after 24 h of seeding into the plates. The fourth-passage non-β-catenin expression MSCs were divided into eight groups: control group, 5-aza, salB, CLM, 5-aza + salB, 5-aza + CLM, salB + CLM, and 5-aza + salB + CLM. The gene and protein expression of cTnT, α-actin, β-myosin, β-catenin, and GSK-3β were detected by quantitative real-time PCR and Western blotting. Our results showed that cTnT expression in 5-aza + salB + CLM group was ninefold higher than in the control group in the non-β-catenin MSCs model, implying that cardiomyocytes differentiation from MSCs is an extremely complicated process and it is necessary to consider the internal and external environmental conditions, such as suitable pharmaceutical inducers, cardiomyocytes microenvironments, inhibition of the negative signaling pathway and so on.     

Seasonal changes in the efflux of sugars from parenchyma cells into the apoplast in poplar stems (Populus × canademis “robusta”)

Summary The efflux of sugars from parenchyma cells into the apoplast has been studied in the wood of Populus × canadensis robusta in relation to the sugar level of the tissue and the sugar content of the tracheal sap during all physiological stages of the year. In poplar, the spring maximum in sugar content of the tracheal sap is clearly not the result of continuous exosmosis during winter but is reached within a short period in spring. The seasonal patterns of starch and sugar content of the wood and of the sugar content of the tracheal sap are described. The efflux of sugars from tissue sections changes drastically over the year, i.e., from 0.3 to 8.3 g mg^–1DWT day^–1. In general, it is high in fall and winter, and low during late spring and summer. However, high tissue sugar levels proved not always to be correlated with high efflux rates or with high sugar levels in the tracheal sap, indicating that the intracellular compartmentalization of sugars, their passive and catalysed release into, and their re-uptake from the apoplast are all essentially involved in determining the actual sugar content of the sap. Sucrose, which is the dominant sugar in the tracheal sap during winter (pH 7.0–7.5) and in the efflux experiments at pH 7.5, in contrast to the hexoses which prevail in the spring sap (pH around 5.5) and also in the efflux experiments at pH 5.6, is considered to be preferentially released in poplar and to become extraplasmatically hydrolysed. The reasons for tree-specific differences are discussed.     

Kinetics of Δψ-Dependent Sucrose Transport in Plasma Membrane Vesicles from Higher Plant Cells

The inside-out vesicles of plasma membranes were isolated from pumpkin stem cells, and the kinetics of sucrose efflux induced by the K⁺-diffusion potential (_D) was studied by measuring light transmission. Two phases differing in their rates and duration were identified in _D-dependent changes of light transmission. The increase in _Delevated the rate and magnitude of the fast phase in light transmission changes but did not markedly affect the rate of the slow phase. These two phases also differed in their sensitivity to inhibitors and to changes in sucrose concentration in the external medium. Measurements of _Dduring sucrose transport by means of the fluorescence probe dis-C₃-(5) revealed differences in the magnitude of _Dand its stability in vesicles loaded with sucrose and mannitol, as well as under the action of inhibitors. The two-phase dependence of sucrose efflux from vesicles on the applied diffusion potential is discussed in the context of modern concepts on the functioning of sucrose carriers in the membranes.     

Fusarium mycotoxins in forage maize — Detection and evaluation

The deoxynivalenol concentrations found in forage maize ranged between 0.24 and 14.29 mg/kg DM (detected by ELISA). When highly contaminated samples were analysed for deoxynivalenol by HPLC or LC-MS the resulting concentrations were in the mean about 50% lower. Furthermore, using LC-MS other type-A and type-B trichothecenes, zearalenone and α-zearalenol were found in these samples. The differences between ELISA and HPLC/LC-MS data for deoxynivalenol are assumed to result from cross-reactions of other trichothecenes with the antibodies used in ELISA and toxin losses from sample purification procedures needed for HPLC and LC-MS analysis. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

Buffer-stimulated citrate efflux in Penicillium simplicissimum: an alternative charge balancing ion flow in case of reduced proton backflow?

Organic acids excreted by filamentous fungi may be used to win metals from industrial secondary raw materials. For a future commercial use a high production rate of organic acids is necessary. The conditions under which the commercially used fungus Aspergillus niger excretes high amounts of citric acid can not be maintained in metal leaching processes. However, Penicillium simplicissimum showed an enhanced citric acid efflux in the presence of an industrial filter dust containing 50% zinc oxide. Because Good buffers of high molarity were able to mimic the effect of zinc oxide, the high buffering capacity of zinc oxide and not an effect of the zinc ions was held responsible for the enhanced citric acid efflux. The presence of ammonium and trace elements reduced this buffer-stimulated citric acid efflux, whereas the plant hormone auxine canceled this reduction. This citric acid efflux was influenced by a depolarization of the membrane: the freely permeable compound tetraphenylphosphoniumbromide decreased the citric acid efflux, without decreasing intracellular citric acid or consumption of glucose and oxygen. Vanadate, an inhibitor of the plasma membrane H⁺-ATPase also reduced the buffer-stimulated citric acid efflux. The role of the efflux of citrate anions as an alternative charge balancing ion flow in case of impaired backflow of extruded protons because of a high extracellular buffering capacity is discussed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - delta electrochemical potential gradient - DES diethylstilbestrol - DMSO dimethyl sulfoxide - TAPS N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid - TEA triethanolamine - TFP trifluoperazine - TPP tetraphenylphosphonium bromide