Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter^low phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.     

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-β and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

Chemical modification of active sites in relation to the catalytic mechanism of F1

Recent studies of chemically modified F₁-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F₁-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F₁ possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the ₃₃ or ₃ moiety relative to the remainder of the F₀F₁ complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles.     

Purification and Characterization of Escherichia coli MreB Protein

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm.     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the ΔyhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in γ-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.     

豚鼠耳蜗中ATP对一氧化氮/环磷酸鸟苷途径的激活作用

实验研究了豚鼠耳蜗中ATP和一氧化氮／环磷酸鸟苷途径(nitric oxide／cyclic guanosine monophosphate,NO／cGMP pathway)的关系。将40只耳廓反射灵敏的健康白色豚鼠随机分为5组,分别对其离体的耳蜗即刻灌流人工外淋巴基础液(artificial perilymph basic solution,APBS)以及溶于人工外淋巴基础液的ATP、一氧化氮合酶抑制剂左旋-N^G-硝基精氨酸(L-N^G-nitroarginine,L-NNA) ATP、可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]草酸重氮[4,3-a]喹恶啉(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one,ODQ) ATP和A-23187(Ca^2 载体),收集耳蜗组织标本,利用放射免疫方法测定耳蜗组织中的cGMP的平均含量,比较各组之间耳蜗组织cGMF平均含量的差异。试验结果显示,向刚离体的耳蜗中灌流ATP和A-23187可以引起耳蜗组织中的cGMP含量升高,而灌流L-NNA和ODQ则可以抑制ATP所引起的耳蜗组织中cGMP含量的升高,提示在耳蜗组织中ATP可以通过升高细胞内Ca^2 浓度的作用而激活NO／cGMF途径。从本实验结果可以提出假说：耳蜗中ATP从神经末梢释放,通过提高细胞内Ca^2 的浓度,有激活NO／cGMP途径的作用,而NO／cGMP又能对ATP进行负反馈调节,两者共同调节耳蜗的生理功能,在耳蜗中存在ATP／Ca^2 -NO／cGMP通路。     

Human erythrocyte flickering: temperature,ATP concentration,water transport,and cell aging,plus a computer simulation

Images of human erythrocytes from a healthy donor were recorded under differential interference contrast (DIC) microscopy; they were acquired rapidly (~336 Hz) and the intensity of the centermost pixel of each cell was recorded for ~60 s (20,000 values). Various techniques were used to analyze the data, including detrended fluctuation analysis (DFA) and multiscale entropy (MSE); however, power spectrum analysis was deemed the most appropriate for metrifying and comparing results. This analysis was used to compare cells from young and old populations, and after perturbing normal conditions, with changes in temperature, adenosine triphosphate (ATP) concentration (using NaF, an inhibitor of glycolysis, and α-toxin, a pore-forming molecule used to permeabilize red cells to ATP), and water transport rates [using glycerol, and p-chloromercuriphenylsulfonic acid (pCMBS) to inhibit aquaporins, AQPs]. There were measurable differences in the membrane fluctuation characteristics in populations of young and old cells, but there was no significant change in the flickering time series on changing the temperature of an individual cell, by depleting it of ATP, or by competing with the minor water exchange pathway via AQP3 using glycerol. However, pCMBS, which inhibits AQP1, the major water exchange pathway, inhibited flickering in all cells, and yet it was restored by the membrane intercalating species dibutyl phthalate (DBP). We developed a computer model to simulate acquired displacement spectral time courses and to evaluate various methods of data analysis, and showed how the flexibility of the membrane, as defined in the model, affects the flickering time course.