Blue light effects on stomata are mediated by the guard cell plasma membrane redox system distinct from the proton translocating ATPase

Abstract. The effects of blue light on stomata are critically analysed. Blue-light-induced increase in stomatal conductance is preceded by membrane hyperpolarization, proton efflux, potassium uptake and malate synthesis in guard cells. Hypothetically, a flavin containing plasma membrane redox system can pump protons out of guard cells on illumination with blue light. It is proposed that this electrogenic proton pump requires NAD(P)H but does not involve ATP/ATPase.     

Cell communication with the neural plate is required for induction of neural markers by BMP inhibition: evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.     

酸雨胁迫下,稀土元素对菠菜膜保护系统作用

利用盆栽实验,探讨了酸雨胁迫下对菠菜膜保护酶系统的防护效应,实验结果表明,单一酸雨处理会造成超氧化物歧化酶（ＳＯＤ）,过氧化酶（ＣＡＴ）活性总体水平下降,其变化曲线呈“Ａ”形,并使过氧化物酶（ＰＯＤ）活性明显增加,施用稀土元素后酸雨胁迫下的植株叶片中ＳＯＤ,ＣＡＴ活性总体水平上升,变化曲线的峰值向酸度较大的方向移动,ＰＯＤ活性上升幅度减小,３种膜保护酶的活性与单一酸雨处理组相比,处在一种结稳定的状     

Electro‐mediated gene transfer and expression are controlled by the life‐time of DNA/membrane complex formation

Background

Electroporation is a physical method used to transfer molecules into cells and tissues. Clinical applications have been developed for antitumor drug delivery. Clinical trials of gene electrotransfer are under investigation. However, knowledge about how DNA enters cells is not complete. By contrast to small molecules that have direct access to the cytoplasm, DNA forms a long lived complex with the plasma membrane and is transferred into the cytoplasm with a considerable delay.

Methods

To increase our understanding of the key step of DNA/membrane complex formation, we investigated the dependence of DNA/membrane interaction and gene expression on electric pulse polarity and repetition frequency.

Results

We observed that both are affected by reversing the polarity and by increasing the repetition frequency of pulses. The results obtained in the present study reveal the existence of two classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression.

Conclusions

The life‐time of DNA/membrane complex formation is of the order of 1 s and has to be taken into account to improve protocols of electro‐mediated gene delivery. Copyright © 2009 John Wiley & Sons, Ltd.     

Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase

This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (?Ψ_m) in living cells and tissues at low values (100-140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ?Ψ_m values (180-220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ?Ψ_m from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ?Ψ_m is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ?Ψ_m was found. From the data we conclude that high matrix ATP/ADP ratios keep ?Ψ_m at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ?Ψ_m-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ?Ψ_m according to the Mitchell Theory.     

A new in vitro system to study the effect of liquid phase turnover and pH on microbial fermentation of concentrate diets for ruminants

A simple and inexpensive in vitro feed evaluation system which estimates microbial fermentation by measuring gas production and allows for adjustment of liquid dilution rate has been design. Working conditions, in terms of substrate and inoculum proportions (experiment 1, EXP 1) and the liquid replacement schedule (experiment 2, EXP 2) were initially set. In EXP 1, three substrate concentrations (10, 20 and 30 mg/ml: S1, S2 and S3) and three inoculum proportions (0.10, 0.20 and 0.30; I10, I20 and I30) were assayed in a 3 × 3 factorial design, in three incubations of 24 h. Gas production (ml/g organic matter) with S1 was the highest, avoiding to be in excess for microbial colonisation and activity and inoculum I20 limited inoculum contribution by self-fermentation; they were hence considered for further trials. In EXP 2, two liquid replacement schedules (every hour from 0 to 12 h and every 3 h from 12 to 24 h, HF, and every 2 h from 0 to 12 h and every 4 h from 12 to 24 h, LF) were compared with a non-replacement (NR) treatment, using three bottles per treatment. No differences among treatments were detected, and thus the LF schedule was chosen because it was easier to carry out. Once these working conditions were adjusted in EXP 1 and 2, the effect of fluid turnover and incubation pH on the extent of microbial fermentation in a mixed diet (experiment 3, EXP 3) was studied in two incubations. Two liquid turnover rates (0.06 and 0.10 h⁻¹) were compared with a non-replacement treatment at two different pH (6.8 and 6.1) in a 3 × 2 factorial design. Liquid outflow increased gas production, dry matter disappearance (DMd) and total volatile fatty acid (VFA) concentration at both pH (P>0.01), but protozoal numbers were unaffected. Liquid replacement at pH 6.8 did not affect the VFA profile, but the acetate proportion diminished (P=0.04) and the propionate increased (P<0.001), with replacement at pH 6.1. Increasing the fluid turnover rate from 0.06 to 0.10 h⁻¹ reduced gas production from 8 to 20 h of incubation (P<0.05) but it did not alter the VFA profile. The volume of gas produced decreased (P<0.05) with incubation pH, as did DMd (P=0.04) and protozoal (P=0.03) and total VFA (P=0.002) concentration. The acetate proportion was lower (P<0.001) and that of propionate and butyrate was higher (P<0.001) at pH 6.1 than at pH 6.8. Both liquid turnover and pH must be taken into consideration for in vitro evaluation of mixed and concentrate diets for ruminants and this system provides a simple and inexpensive method for doing this.     

Ethanol addition on inactivation of Saccharomyces pastorianus by a two‐stage system with low‐pressure carbon dioxide microbubbles can accelerate the cell membrane injury

The effect of ethanol on the inactivation of Saccharomyces pastorianus by a two‐stage system with low‐pressure carbon dioxide microbubbles (two‐stage MBCO₂) was investigated. Zero and >5 log reductions of S. pastorianus populations suspended in physiological saline (PS) containing 0% and 10% ethanol, respectively, occurred by the two‐stage MBCO₂ at a mixing vessel pressure of 1 MPa and a heating coil temperature of 40°C. Conversely, the detected number of surviving S. pastorianus cells in PS containing 5% ethanol was higher in yeast and mold agar (YMA, an optimum agar) than YMA with 2.5% sodium chloride, followed by yeast nitrogen base agar (YNBA, a minimum agar). The fluorescence polarization of S. pastorianus in PS containing 5% and 10% ethanol increased similarly with exposure time in the heating coil of two‐stage MBCO₂ and was correlated with the surviving cell number measured in YNBA. The intracellular pH (pH_in) of S. pastorianus in PS containing 5% ethanol decreased linearly with exposure time in the heating coil of two‐stage MBCO₂. Also, the pH_in‐lowering of S. pastorianus in PS containing 10% ethanol was drastically caused by two‐stage MBCO₂ at 1 min exposure time in the heating coil but then stayed constant until 5 min, agreeing with the inactivation efficiency. Therefore, ethanol in S. pastorianus suspension was suggested to accelerate the cell membrane injury caused by two‐stage MBCO₂. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:282–286, 2018     

The combination effects of phenolic compounds and fluconazole on the formation of ergosterol in Candida albicans determined by high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS) with atmospheric pressure chemical ionization (APCI) for the quantification of ergosterol, lanosterol, and squalene was developed to evaluate the combination effects of phenolic compounds with fluconazole on ergosterol biosynthesis in Candida albicans. The three analytes were separated by a column of C18 and were quantified without interference with each other using positive mode tandem mass spectrometry (MS/MS). Molecular ions of ergosterol and lanosterol were detected as the [M+H-H2O]+ ion species at m/z 380 and 410, whereas squalene appeared as the [M+H]+ ion species at m/z 412. On fragmentation of ergosterol, lanosterol, and squalene, the product ions at m/z 69, 149, and 109, respectively, were present as major fragments. These product ions were used for the quantification of them in multiple reaction monitoring acquisition mode. The relationship between signal intensity and the analytes' concentration was linear over the concentration range of 0.05-10 microg/ml. Following the treatment of C. albicans with fluconazole in combination with albicanyl caffeate, resveratrol, and 3,4'-difluorostilbene, respectively, the content of ergosterol in both the sensitive and resistant C. albicans showed depletion, whereas the squalene showed accumulation especially in the sensitive isolates determined with the method developed.     

A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR

We have developed an online analytical method that combines A431 cell membrane chromatography (A431/CMC) with high performance liquid chromatography and mass spectrometry (LC/MS) for identifying active components from Radix Caulophylli acting on human EGFR. Retention fractions on A431/CMC model were captured onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using Sorafenib tosylate as a positive control, taspine and caulophine from Radix Caulophylli were identified as the active molecules which could act on the EGFR. This A431/CMC-online-LC/MS method can be applied for screening active components acting on EGFR from traditional Chinese medicines exemplified by Radix Caulophylli and will be of great utility in drug discovery using natural medicinal herbs as a source of novel compounds.     

Regulation of the cancer cell membrane lipid composition by NaCHOleate : Effects on cell signaling and therapeutical relevance in glioma

This review summarizes the cellular bases of the effects of NaCHOleate (2-hydroxyoleic acid; 2OHOA; Minerval) against glioma and other types of tumors. NaCHOleate, activates sphingomyelin synthase (SGMS) increasing the levels of cell membrane sphingomyelin (SM) and diacylglycerol (DAG) together with reductions of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The increases in the membrane levels of NaCHOleate itself and of DAG induce a translocation and overexpression of protein kinase C (PKC) and subsequent reductions of Cyclin D, cyclin-dependent kinases 4 and 6 (CDKs 4 and 6), hypophosphorylation of the retinoblastoma protein, inhibition of E2F1 and knockdown of dihydrofolate reductase (DHFR) impairing DNA synthesis. In addition in some cancer cells, the increases in SM are associated with Fas receptor (FasR) capping and ligand-free induction of apoptosis. In glioma cell lines, the increases in SM are associated with the inhibition of the Ras/MAPK and PI3K/Akt pathways, in association with p27Kip1 overexpression. Finally, an analysis of the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database for glioma patient survival shows that the weight of SM-related metabolism gene expression in glioma patients' survival is similar to glioma-related genes. Due to its low toxicity and anti-tumoral effect in cell and animal models its status as an orphan drug for glioma treatment by the European Medicines Agency (EMA) was recently acknowledged and a phase 1/2A open label, non-randomized study was started in patients with advanced solid tumors including malignant glioma. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

亚抑菌浓度博落回生物碱对ExPEC主要外膜蛋白和II型T-A系统表达的影响

【背景】血根碱、白屈菜红碱、原阿片碱等生物碱是我国二类新兽药博落回散和博普总碱散的主要成分,具有广泛的药理作用。【目的】研究亚抑菌浓度血根碱、白屈菜红碱、原阿片碱对肠外致病性大肠杆菌(extraintestinal pathogenic Escherichia coli,ExPEC)主要外膜蛋白及其调控基因和Ⅱ型毒素-抗毒素(toxin-antitoxin,T-A)系统基因表达的影响,初步探讨博落回生物碱对ExPEC细菌生理活动影响的可能机制。【方法】比较不同Ⅱ型T-A系统基因yafON、hicAB、prlF-yhaV缺失的ExPEC对血根碱、白屈菜红碱、原阿片碱及抗生素的最小抑菌浓度;在1/2MIC亚抑菌浓度血根碱、白屈菜红碱、原阿片碱条件下,比较它们对ExPEC野生型(wild type,WT)菌株和外膜蛋白tolC缺失菌株(ΔtolC)的不同外膜蛋白基因ompC、ompX、tolC、ompF和调控基因acrR、rob、marR、rpoS、soxS表达的影响,以及对T-A系统基因yafON、hicAB、prlF-yhaV表达的影响。【结果】T-A系统hicAB和prlF-yhaV缺...