Redox Transfer across the Inner Chloroplast Envelope Membrane

In leaves of spinach plants (Spinacia oleracea L.) grown in ambient CO₂ the subcellular contents of adenylates, pyridine nucleotides, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, glutamate, 2-oxoglutarate, and aspartate were assayed in the light and in the dark by nonaqueous fractionation technique. From the concentrations of NADP and NADPH determined in the chloroplast fraction of illuminated leaves the stromal NADPH to NADP ratio is calculated to be 0.5. For the cytosol a NADH to NAD ratio of 10⁻³ is calculated from the assay of the concentrations of NAD, malate, glutamate, aspartate, and 2-oxoglutarate on the assumption that the reactions catalyzed by the cytosolic glutamate oxaloacetate transaminase and malate dehydrogenase are not far away from equilibrium. For the transfer of redox equivalents from the chloroplastic NADPH to the cytosolic NAD two metabolite shuttles are operating across the inner envelope membrane: the triosephosphate-3-phosphoglycerate shuttle and the malate-oxaloacetate shuttle. Although both shuttles would have the capacity to level the redox state of the stromal and cytosolic compartment, this apparently does not occur. To gain an insight into the regulatory processes we calculated the free energy of the enzymic reactions and of the translocation steps involved. From the results it is concluded that the triosephosphate-3-phosphoglycerate shuttle is mainly controlled by the chloroplastic reaction of 3-phosphoglycerate reduction and of the cytosolic reaction of triosephosphate oxidation. The malate-oxaloacetate shuttle is found to be regulated by the chloroplastic NADP-malate dehydrogenase and also by the translocating step across the envelope membrane.     

Thermolability of the Alternative Electron Transport Pathway in Higher Plant Mitochondria

Mitochondria from four plant species showing normal (Arum maculatum L., Arum italicum Mill., Sauromatum guttatum Schott) or induced (Solanum tuberosum L.) resistance to cyanide were submitted to temperature treatments up to 90 min at 45°C. The activity of the alternative, cyanide-resistant electron transport pathway was specifically and deeply altered by temperature treatments. Hydrogen sulfide was released in direct proportion to the reduction of activity of the alternative pathway. Only a small fraction (? 20%) of the total labile sulfide content of the mitochondria was associated with the operation of this pathway. In cyanide-resistant mitochondria, the cytochrome pathway was much more resistant to thermal inactivation than the alternative pathway. On the contrary, in cyanide-sensitive mitochondria (with no alternative pathway) the cytochrome pathway was highly sensitive to temperature treatments. These results indicate that the presence of a cyanide-resistant alternative pathway is correlated with a higher degree of resistance to thermal denaturation of the cytochrome pathway. They also strongly suggest that iron-sulfur proteins are regular components of the alternative pathway.     

始于质体/叶绿体的ABA信号通路

该文全面评述了植物激素脱落酸（ABA）受体的研究进展概况,重点介绍细胞内ABA受体ABAR/镁螯合酶H亚基CHLH对ABA信号感知和向下游转导的研究进展,总结了ABAR介导的、起始于质体/叶绿体的ABA信号通路。ABAR是一个跨越叶绿体被膜的蛋白质,其N-端和C-端暴露在细胞质中;ABAR在细胞质一侧的C-端部分与一组WRKY转录因子（WRKY18、WRKY40、WRKY60）相互作用。WRKY18、WRKY40和WRKY60是一组转录抑制因子。它们互相协作,抑制下游重要的ABA信号调节子基因（如ABI4、ABI5、ABF4和MYB2等）的表达,从而负调节ABA信号通路。WRKY40是其中的核心调节子,WRKY18协助加强WRKY40对ABA信号的负调节。ABAR与ABA信号分子结合后,可以刺激WRKY40从细胞核转移至细胞质,促进ABAR与WRKY40的相互作用;进而激发一种未知因子（或信号系统）,阻遏WRKY40的表达,从而解除WRKY40对ABA响应基因转录的抑制,最终实现ABA的生理效应。这些发现描述了一个从信号原初识别到下游基因表达的新的ABA信号通路。论文最后对未来该领域的研究方向进行了讨论。     

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C₁₈ fatty acids.     

Electron Transfer Pathways and Dynamics of Chloroplast NADPH-dependent Thioredoxin Reductase C (NTRC)

NADPH-dependent thioredoxin reductases (NTRs) contain a flavin cofactor and a disulfide as redox-active groups. The catalytic mechanism of standard NTR involves a large conformational change between two configurations. Oxygenic photosynthetic organisms possess a plastid-localized NTR, called NTRC, with a thioredoxin module fused at the C terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs) and thus is involved in the protection against oxidative stress, among other functions. Although the mechanism of electron transfer of canonical NTRs is well established, it is not yet known in NTRC. By employing stopped-flow spectroscopy, we have carried out a comparative kinetic study of the electron transfer reactions involving NTRC, the truncated NTR module of NTRC, and NTRB, a canonical plant NTR. Whereas the three NTRs maintain the conformational change associated with the reductive cycle of catalysis, NTRC intramolecular electron transfer to the thioredoxin module presents two kinetic components (k_ET of ∼2 and 0.1 s⁻¹), indicating the occurrence of additional dynamic motions. Moreover, the dynamic features associated with the electron transfer to the thioredoxin module are altered in the presence of 2-Cys Prx. NTRC shows structural constraints that may locate the thioredoxin module in positions with different efficiencies for electron transfer, the presence of 2-Cys Prx shifting the conformational equilibrium of the thioredoxin module to a specific position, which is not the most efficient.     

Effect of the Pool Size of Stromal Reductants on the Alternative Pathway of Electron Transfer to Photosystem I in Chloroplasts of Intact Leaves

The effect of elevated temperature on electron flow to plastoquinone pool and to PSI from sources alternative to PSII was studied in barley (Hordeum vulgare L.) and maize (Zea mays L.) leaves. Alternative electron flow was characterized by measuring variable fluorescence of chlorophyll and absorption changes at 830 nm that reflect redox changes of P700, the primary electron donor of PSI. The treatment of leaves with elevated temperature resulted in a transient increase in variable fluorescence after cessation of actinic light. This increase was absent in leaves treated with methyl viologen (MV). The kinetics of P700⁺ reduction in barley and maize leaves treated with DCMU and MV exhibited two exponential components. The rate of both components markedly increased with temperature of the heat pretreatment of leaves when the reduction of P700⁺ was measured after short (1 s) illumination of leaves. The acceleration of both kinetic components of P700⁺ reduction by high-temperature treatment was much less pronounced when P700⁺ reduction rate was measured after illumination of leaves for 1 min. Since the treatment of leaves with DCMU and MV inhibited both the electron flow to PSI from PSII and ferredoxin-dependent cycling of electrons around PSI, the accelerated reduction of P700⁺ indicated that high temperature treatment activated electron flow to PSII from reductants localized in the chloroplast stroma. We conclude that the lesser extent of activation of this process by elevated temperature after prolonged illumination of heat-inhibited leaves is caused by depletion of the pool stromal reductants in light due to photoinduced electron transfer from these reductants to oxygen.     

北京科学家揭示MicroRNA途径介导的DNA甲基化

<正>北京中关村生命科学园国立生物科学研究所和北京师范大学新乡医学院生命科学院的研究人员共同研究,报道了一类新的MicroRNA(long miRNAs)途径介导的DNA甲基化作用。这一研究成果5月14日发表在《MolecularCell》最新一期杂志上。     

24p3/NGAL介导的铁转运途径

生物体内存在另一转铁途径。脂笼蛋白(lipocalin)家族的成员24p3／NGAL介导铁向细胞内转运,并在具有酸性环境的核内体(endosome)中与铁解离,进而调节铁蛋白(ferritin,Fn)基因和转铁蛋白受体-1(transferrin receptor-1,TfR-1)基因的表达。24p3／NGA转铁途径在亚细胞水平上与转铁蛋白(tansferrin,Tf)类似但相互独立。在胚肾发育过程中,24p3／NGAL与Tf介导的铁转运途径为不同时期的原始肾上皮细胞生长与分化所必需。深入研究24p3／NGAL转铁途径的分子机制及与Tf的异同有十分重要的意义。     

An Alternative Pathway Mediates the Mouse and Human Cone Visual Cycle

One of the fundamental mysteries of the human visual system is the continuous function of cone photoreceptors in bright daylight. As visual pigment is destroyed, or bleached, by light [1], cones require its rapid regeneration, which in turn involves rapid recycling of the pigment's chromophore. The canonical visual cycle for rod and cone pigments involves recycling of their chromophore from all-trans retinol to 11-cis retinal in the pigment epithelium, adjacent to photoreceptors [2]. However, shortcomings of this pathway indicate the function of a second, cone-specific, mechanism for chromophore recycling [3]. Indeed, biochemical [3], [4], [5], [6] and [7] and physiological [8] studies on lower species have described a cone-specific visual cycle in addition to the long-known pigment epithelium pathway. Two important questions remain, however: what is the role of this pathway in the function of mammalian cones, and is it present in higher mammals, including humans? Here, we show that mouse, primate, and human neural retinas promote pigment regeneration and dark adaptation selectively in cones, but not in rods. This pathway supports rapid dark adaptation of mammalian cones and extends their dynamic range in background light independently of the pigment epithelium. This pigment-regeneration mechanism is essential for our daytime vision and appears to be evolutionarily conserved.     

Alternative Complement Pathway in Schizophrenia

In the present study, we evaluated functional activity of the alternative pathway of complement in schizophrenia by measuring the alternative pathway hemolytic activity (AH50) of complement as well as hemolytic activity of the complement C3 component (C3H50) in the blood of patients with schizophrenia and healthy subjects. To assess the influence of neuroleptic treatment on measured parameters, both drug-free and medicated patients were examined. In addition, correlation analysis between AH50 and C3H50 has been performed. The results of the present study clearly demonstrate upregulation of the alternative complement cascade in schizophrenia and activator effect of neuroleptics on complement alternative pathway. Based upon the results obtained we hypothesize that hyperactivation of the alternative complement pathway in schizophrenia is stimulated by apoptotic cells.     

Stimulation of the Alternative Pathway by Succinate and Malate

Stimulation of the cyanide-resistant oxidation of exogenous NADH in potato (Solanum tuberosum L. cv Bintje) tuber callus mitochondria was obtained with succinate, malate, and pyruvate. Half-maximal stimulation was observed at a succinate or malate concentration of 3 to 4 mM, which is considerably higher than that found for pyruvate (0.128 mM). No effect of succinate or malate addition was found when duroquinone was the electron acceptor. Duroquinol oxidation via the alternative pathway was poor and not stimulated by organic acids. Under stimulating conditions, no swelling or contraction of the mitochondria could be observed. Conversely, variation of the osmolarity did not affect the extent of stimulation. However, the assay temperature had a significant effect: no stimulation occurred at temperatures below 16 to 20[deg]C. Membrane fluidity measurements showed a phase transition at about 17[deg]C. Ubiquinone reduction levels were not significantly higher in the presence of succinate and malate, but the kinetics of the alternative oxidase were changed in a way comparable to that found for stimulation by pyruvate. At low temperatures the alternative oxidase displayed "activated" kinetics, and a role for membrane fluidity in the stimulation of the alternative pathway by carboxylic acids is suggested.     

Fast-activating Channel Controls Cation Fluxes across the Native Chloroplast Envelope

A prerequisite for photosynthetic CO(2) fixation is the maintenance of alkaline pH in the stroma. This is achieved by H(+) pumping from the stroma to the cytosol, electrically balanced by an influx of cations through some unidentified non-selective envelope channels. In this study, the patch-clamp technique was applied to isolated Pisum sativum L. (pea) chloroplasts, and a fast-activating chloroplast cation (FACC) channel was discovered in the native envelope. This channel opens within a few milliseconds upon voltage steps to large positive or negative potentials. Remarkably, the single-channel conductance increased fivefold, from approximately 40 pS to approximately 200 pS (symmetric 250 mM KCl), upon a potential change from zero to +/- 200 mV. The FACC channel conducts all physiologically essential inorganic cations (K(+), Na(+), Ca(2+), Mg(2+)) with little preference. An increase of stromal pH from 7.3 to 8.0, mimicking dark-light transition, caused about a 2-fold decrease of the FACC channel activity within a physiologically relevant potential range. The FACC channel was completely and irreversibly blocked by Gd(3+). Based on the estimated transport capacity of the whole chloroplast population of FACC channels together with the envelope H(+)-ATPases, these channels can mediate electroneutral K(+)/H(+) exchange across the envelope, enabling stroma alkalinization, thereby allowing an optimal photosynthetic performance.     

Interaction of Ribosomes and the Cell Envelope of Escherichia coli Mediated by Lysozyme

Evidence is presented suggesting the existence of a natural ribosome-membrane complex. A reconstruction system is described wherein free ribosomes form a complex which appears to involve cell fragments. The reconstructed complex is similar in stability to the inferred natural complex. The reconstructed complex is generated by lysozyme, and it is concluded that at least part of the inferred natural complex is also generated by lysozyme. These results are discussed with reference to existing data concerning certain membrane-associated systems in bacteria.     

Essential Role of VIPP1 in Chloroplast Envelope Maintenance in Arabidopsis

VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), proposed to play a role in thylakoid biogenesis, is conserved in photosynthetic organisms and is closely related to Phage Shock Protein A (PspA), which is involved in plasma membrane integrity in Escherichia coli. This study showed that chloroplasts/plastids in Arabidopsis thaliana vipp1 knockdown and knockout mutants exhibit a unique morphology, forming balloon-like structures. This altered morphology, as well as lethality of vipp1, was complemented by expression of VIPP1 fused to green fluorescent protein (VIPP1-GFP). Several lines of evidence show that the balloon chloroplasts result from chloroplast swelling related to osmotic stress, implicating VIPP1 in the maintenance of plastid envelopes. In support of this, Arabidopsis VIPP1 rescued defective proton leakage in an E. coli pspA mutant. Microscopy observation of VIPP1-GFP in transgenic Arabidopsis revealed that VIPP1 forms large macrostructures that are integrated into various morphologies along the envelopes. Furthermore, live imaging revealed that VIPP1-GFP is highly mobile when chloroplasts are subjected to osmotic stress. VIPP1-GFP showed dynamic movement in the transparent area of spherical chloroplasts, as the fluorescent molecules formed filament-like structures likely derived from disassembly of the large VIPP1 complex. Collectively, our data demonstrate that VIPP1 is a multifunctional protein in chloroplasts that is critically important for envelope maintenance.     

Metformin Plus Caloric Restriction Show Anti-epileptic Effects Mediated by mTOR Pathway Inhibition

Caloric restriction (CR) has anti-epileptic effects in different animal models, at least partially due to inhibition of the mechanistic or mammalian target of rapamycin (mTOR) signaling pathway. Adenosine monophosphate-activated protein kinase (AMPK) inhibits mTOR cascade function if energy levels are low. Since hyper-activation of mTOR participates in epilepsy, its inhibition results in beneficial anti-convulsive effects. A way to attain this is to activate AMPK with metformin. The effects of metformin, alone or combined with CR, on the electrical kindling epilepsy model and the mTOR cascade in the hippocampus and the neocortex were studied. Combined metformin plus CR beneficially affected many kindling aspects, especially those relating to generalized convulsive seizures. Therefore, metformin plus CR could decrease measures of epileptic activity in patients with generalized convulsive seizures. Patients that are obese, overweight or that have metabolic syndrome in addition to having an epileptic disease are an ideal population for clinical trials to test the effectiveness of metformin plus CR.