饲养层FGF2表达量对猕猴胚胎干细胞自我更新及多能性的影响

用转基因和RNA干扰(RNAi)法建立5组不同成纤维细胞生长因子-2(fibroblast growth factor-2,FGF2)表达量的猕猴耳部皮肤成纤维细胞(MESF)系:过表达FGF2组(f1),过表达的阴性对照组(f2),FGF2 RNA干扰组(f3),RNA干扰的阴性对照组(f4)和未作任何处理的对照组(f5).5组MESF的FGF2表达量相对值为f1:f2:f3:f4:f5=4:2:1:2:2;c-fos,TGF-β1,INHBA,Gremlinl在f1中表达量上升,在f3中表达量下降;BMP4,TGF-β2在f1中表达量下降,在f3中表达量上升;表明内源FGF2能够作用于MESF的TGF-β信号通路,引起相关基因表达量的变化.用这砦细胞作为饲养层长期培养(10代)猕猴胚胎干细胞(RhESC),结果在f1上培养的RhESC增殖速度都比对照组快,并且c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达量均上升,BMP4表达下调;在f3上培养的RhESC增殖较慢,BMP4表达上调,c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达下调.5组MESF上培养的RhESC形成的EB均表达各胚层早期标记基因(marker),说明RhESC的多能性没有受到影响,但表达量有差异,f1上RhESC形成的EB所有marker都低表达.结果表明饲养层的FGF2含量不仅影响自身相关基凶的表达,还对RhESC的自我更新有一定的作用.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

Nouvelles C-glycosylflavones extraites de Spergularia rubra

Three 6,8-di-C-glucosylflavones: 6,8-di-C-β-d-glucopyranosylapigenin, luteolin and chrysoeriol were isolated from the whole plant of Spergularia rubra. Two new compounds, 7,2″-di-O-β-d-glucopyranosyl-6-C-α-l-arabinopyranosylapigenin and 6-C-α-l-arabinopyranosylapigenin (isomollupentin), were also characterized. Structural assignments were based on ¹H NMR and MS spectra and on comparison with synthetic samples. MS fragmentation patterns of the new di-O-glucosyl compound PM derivative and of its acid hydrolysis product are given.     

Synthesis of 4-epi-2-deoxy-2-Heq-N-acetylneuraminic acid and 2,4-dideoxy-2-Heq N-acetylneuraminic acid

We have continued our work to develop novel analogues of sialic acid [1–4] that may specifically modulate the interaction between endogenous sialic acid and influenza virus haemagglutinin [3,5,6]. Functional groups of sialic acid that have been implicated for this virus-host recongnition are the glycerol side chain, N-acetyl group and the axially oriented carboxylic acid function In this report we describe the synthesis of two analogues, namely, 4-epi-2-deoxy-2-H_eq-N-acetylneuraminic acid (4-epi-2-d-2-H_eq-Neu5Ac) and 2,4-dideoxy-2-H_eq-N-acetylneuraminic acid (2,4-d₂-2-H_eq-Neu5Ac).     

2-Methylbutyryl CoA dehydrogenase from mitochondria of Ascarissuum and its relationship to NADH-dependent 2-methylcrotonyl CoA reduction

Acyl CoA dehydrogenase and electron-transfer flavoprotein have been isolated and partially purified from mitochondria of the anaerobic nematode, $\text{Ascaris}\text{suum}$. Dehydrogenase activity was greatest with 2-methylbutyryl CoA and the relative substrate specificities of the ascarid dehydrogenase(s) differ greatly from their mammalian counterparts. It appears that the ascarid dehydrogenase functions physiologically as a reductase, catalyzing the final step in the synthesis of branched-chain fatty acids. In fact, incubations of $\text{A}\text{.}\text{suum}$ mitochondrial membranes with electron-transfer flavoprotein, 2-methylbutyryl CoA dehydrogenase, 2-methylcrotonyl CoA and NADH resulted in a substantial, rotenone-sensitive, 2-methylbutyrate synthesis. These results suggest that the ascarid electron-transport chain and at least two soluble mitochondrial proteins are involved in the NADH-dependent reduction of 2-methylcrotonyl CoA.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

Evaluation of the Ca2+ Concentration in Purified Nerve Terminals: Relationship Between Ca2+ Homeostasis and Synaptosomal Preparation

The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin-2 and fura-2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 +/- 2 nM for Percoll (C-band) synaptosomes, loaded at 30 degrees C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37 degrees C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30 degrees or 37 degrees C). Loading quin-2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin-2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura-2 is preferable in synaptosomes, although above a critical fura-2-AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura-2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37 degrees C. This study shows the applicability of measuring steady-state [Ca]i and dynamic [Ca]i changes quantitatively in fura-2-loaded synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of H2O2 in Brain Mitochondria

Generation of H2O2 by rat brain mitochondria using succinate and glycerol-1-phosphate as substrates has been demonstrated. Earlier workers were unable to detect this activity in sucrose-Tris buffer. We found that this was due to a lag in the expression of activity in sucrose medium. Using phosphate buffer (50 mM), good rates are now obtained. Generation of H2O2 by rat brain mitochondria required the presence of antimycin A and was dependent on the substrates succinate and glycerol-1-phosphate. Low rates were obtained with NAD+-linked substrates and none with choline, glutamate, and NADH. The Km and Vmax values for H2O2 generation were considerably lower than the corresponding values for the respective dehydrogenase activity, measured by dye reduction. Oxygen-radical scavengers inhibited H2O2 generation, suggesting oxygen radical involvement. Depletion of ubiquinone from mitochondria resulted in loss of H2O2 generation. Reconstitution of such depleted particles with ubiquinone restored the capacity to generate H2O2 in a concentration-dependent manner. Levels of H2O2 production were found to be maximal in cerebellum. Brain mitochondria from rabbit, hamster, mouse, and guinea pig also have the capacity to generate H2O2 on oxidation of glycerol-1-phosphate.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Preparation of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

The large-scale preparation of a dialkylglycerophosphocholine with unequal alkyl moieties in positions 1 and 2 is reported.     

Root responses along a subambient to elevated CO2 gradient in a C3–C4 grassland

Atmospheric CO₂ (C_a) concentration has increased significantly during the last 20 000 years, and is projected to double this century. Despite the importance of belowground processes in the global carbon cycle, community‐level and single species root responses to rising C_a are not well understood. We measured net community root biomass over 3 years using ingrowth cores in a natural C₃–C₄ grassland exposed to a gradient of C_a from preglacial to future levels (230–550 μmol mol^?1). Root windows and minirhizotron tubes were installed below naturally occurring stands of the C₄ perennial grass Bothriochloa ischaemum and its roots were measured for respiration, carbohydrate concentration, specific root length (SRL), production, and lifespan over 2 years. Community root biomass increased significantly (P<0.05) with C_a over initial conditions, with linear or curvilinear responses depending on sample date. In contrast, B. ischaemum produced significantly more roots at subambient than elevated C_a in minirhizotrons. The lifespan of roots with five or more neighboring roots in minirhizotron windows decreased significantly at high C_a, suggesting that after dense root growth depletes soil resource patches, plants with carbon surpluses readily shed these roots. Root respiration in B. ischaemum showed a curvilinear response to C_a under moist conditions in June 2000, with the lowest rates at C_a<300 μmol mol^?1 and peak activity at 450 μmol mol^?1 in a quadratic model. B. ischaemum roots at subambient C_a had higher SRLs and slightly higher carbohydrate concentrations than those at higher C_a, which may be related to drier soils at low C_a. Our data emphasize that belowground responses of plant communities to C_a can be quite different from those of the individual species, and suggest that complex interactions between and among roots and their immediate soil environment influence the responses of root physiology and lifespan to changing C_a.     

Photophosphorylation in Scenedesmus in vivo: O2 evolution, ATP pools and transients, and phosphate binding during photoreduction of NO3−, NO2− and CO2

The relation between light-induced electron transport with NO₃^?, NO₂^? or CO₂ as acceptors, ATP pools and transients in dark-light-dark transitions, and phosphate uptake was examined in phosphorus-starved cells of Scenedesmus obtusiusculus Chod. Net O₂ evolution at saturating light was around 6 μmol × (mg chlorophyll × h)^?1 in the absence of any acceptor, but reached average rates of 21, 65 and 145 μmol × (mg chlorophyll × h)^?1 upon additions of 5 mM KNO₃, KNO₂ and KHCO₃, respectively. The apparent rate of photophosphorylation in transition experiments was only a few percent of the rate calculated from CO₂-dependent O₂ evolution. Blocking non-cyclic electron transport with DCMU inhibited phosphate assimilation, but acceleration of non-cyclic electron flow by addition of NO₃^? or NO₂^? did not stimulate phosphate assimilation as compared to the situation without an acceptor. A functional non-cyclic system might primarily be needed for an efficient shuttle transfer of ATP from the chloroplast to the cytoplasm. An inhibition of the non-cyclic system due to lack of reducible substrates accelerates the cyclic system and thus indicates a regulation mechanism between the two systems.     

Seasonal CO2 exchange patterns of developing peach (Prunus persica) fruits in response to temperature, light and CO2 concentration

CO₂ exchange rates per unit dry weight, measured in the field on attached fruits of the late-maturing Cal Red peach cultivar, at 1200 μmol photons m^?2S^?1 and in dark, and photosynthetic rates, calculated by the difference between the rates of CO₂ evolution in light and dark, declined over the growing season. Calculated photosynthetic rates per fruit increased over the season with increasing fruit dry matter, but declined in maturing fruits apparently coinciding with the loss of chlorophyll. Slight net fruit photosynthetic rates ranging from 0. 087 ± 0. 06 to 0. 003 ± 0. 05 nmol CO₂ (g dry weight)^?1 S^?1 were measured in midseason under optimal temperature (15 and 20°C) and light (1200 μmol photons m^?2 S^?1) conditions. Calculated fruit photosynthetic rates per unit dry weight increased with increasing temperatures and photon flux densities during fruit development. Dark respiration rates per unit dry weight doubled within a temperature interval of 10°C; the mean seasonal O₁₀ value was 2. 03 between 20 and 30°C. The highest photosynthetic rates were measured at 35°C throughout the growing season. Since dark respiration rates increased at high temperatures to a greater extent than CO₂ exchange rates in light, fruit photosynthesis was apparently stimulated by high internal CO₂ concentrations via CO₂ refixation. At 15°C, fruit photosynthetic rates tended to be saturated at about 600 μmol photons m^?2 S^?1. Young peach fruits responded to increasing ambient CO₂ concentrations with decreasing net CO₂ exchange rates in light, but more mature fruits did not respond to increases in ambient CO₂. Fruit CO₂ exchange rates in the dark remained fairly constant, apparently uninfluenced by ambient CO₂ concentrations during the entire growing season. Calculated fruit photosynthetic rates clearly revealed the difference in CO₂ response of young and mature peach fruits. Photosynthetic rates of younger peach fruits apparently approached saturation at 370 μl CO₂1^?2. In CO₂ free air, fruit photosynthesis was dependent on CO₂ refixation since CO₂ uptake by the fruits from the external atmosphere was not possible. The difference in photosynthetic rates between fruits in CO₂-free air and 370 μl CO₂ 1^?1 indicated that young peach fruits were apparently able to take up CO₂ from the external atmosphere. CO₂ uptake by peach fruits contributed between 28 and 16% to the fruit photosynthetic rate early in the season, whereas photosynthesis in maturing fruits was supplied entirely by CO₂ refixation.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

H2O2诱导的2BS早老细胞中自噬体增多

自噬在细胞复制性衰老中起着重要的作用.然而,早老细胞中的自噬现象基本无相关的报道.本文通过外源性过氧化氢(H₂O₂)的诱导,构建人胚肺二倍体成纤维细胞（2BS细胞）早老模型.首先,通过SA-β-gal染色,验证细胞早老;从形态学和特异标志分子及雷帕霉素作用的靶位点(mTOR)信号通路不同角度检测自噬的变化,其中形态学检测包括丹（磺）酰戊二胺(MDC)自噬分子定量法及电镜自噬超微结构的观察;特异标志分子LC3的检测包括GFP-LC3自噬定位法和免疫印迹法检测LC3;及检测mTOR信号通路下游激酶p70S6蛋白的表达变化.结果表明,过氧化氢诱导的早老细胞中自噬体相对年轻细胞明显增多,且具有保护早老细胞的作用.     

Different Tolerance to Light Stress in NO3−- and NH4+-Grown Phaseolus vulgaris L.

Abstract: NH₄⁺‐grown plants are more sensitive to light stress than NO₃^?‐grown plants, as indicated by reduced growth and intervenal chlorosis of French bean (Phaseolus vulgaris L.). Measuring the time course of F_v/F_m ratios under photoinhibitory light regimes did not reveal any difference in PS II damage between NO₃^?‐ and NH₄⁺‐grown plants, in spite of some indications of higher energy quenching in NO₃^?‐grown plants. Also, a direct action of NH₄⁺ as an uncoupler at the thylakoid membrane could be excluded. Instead, biochemical analysis revealed enhanced lipid peroxidation and higher activity of scavenging enzymes in NH₄⁺‐grown plants indicating that these plants make use of metabolic pathways with stronger radical formation. Evidence for higher rates of photorespiration in NH₄⁺‐grown plants came from experiments showing that electron flux and O₂ evolution were decreased by SHAM in NH₄⁺‐grown plants, and by antimycin A in NO₃^?‐grown plants. Further, the comparison of electron flux and of photoacoustic measurements of O₂ evolution suggested that in NH₄⁺‐grown plants the Mehler reaction was also increased, at least in the induction phase. However, the major cause of N form‐dependent stress sensitivity is assumed to be in the coupling between photosynthesis and respiration, i.e., NO₃^?‐grown plants can utilize the TCA cycle for the generation of C skeletons for amino acid synthesis, thus improving the ATP: reductant balance, whereas NH₄⁺‐grown plants have enhanced rates of photorespiration.