Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Crystal Structure of Monomeric Photosystem II from Thermosynechococcus elongatus at 3.6-? Resolution

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222₁, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q_A, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn₄Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn₄Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.     

中国常见食物ω-3脂肪酸含量

目的：查阅国内外食物成分数据，建立中国常见食物ω 3脂肪酸的含量数据表，为ω-3脂肪酸膳食营养研究提供参考。方法：查找国内外食物数据库中ω 3脂肪酸含量的数据，根据中国居民饮食习惯，整理出中文ω 3脂肪酸食物成分表。结果：经筛选比较，最终收集日本文部科学省于2015年修订的日本食品标准成分表中368种、美国农业部发布于2014年更新于官网的食物成分表中56种和《中国食物成分表2004》中6种食物ω 3脂肪酸的含量。将中国居民常吃的食物按照粮谷类、豆类、蔬菜类、菌藻类、水果类、坚果类、畜肉类、禽肉类、软体类水产、甲壳类水产、鱼类、蛋奶类、油脂类、其他类分列出食物的ALA、EPA和DHA的含量。     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

CP12 from Chlamydomonas reinhardtii, a Permanent Specific ��Chaperone-like�� Protein of Glyceraldehyde-3-phosphate Dehydrogenase

A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A₄ glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific “chaperone-like protein” for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A₂B₂ GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most photosynthetic organisms, including cyanobacteria (1, 2), higher plants (3), the diatom Asterionella formosa (4, 5), and green (1) and red algae (6). It allows the formation of a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH),³ two key enzymes of the Calvin cycle pathway, and was recently shown to interact with fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway (7). The phosphoribulokinase·GAPDH·CP12 complex has been extensively studied in Chlamydomonas reinhardtii (8, 9) and in Arabidopsis thaliana (10, 11). In the green alga C. reinhardtii, the interaction between CP12 and GAPDH is strong (8). GAPDH may exist as a homotetramer composed of four GapA subunits (A₄) in higher plants, cyanobacteria, and green and red algae (6, 12), but in higher plants, it can also exist as a heterotetramer (A₂B₂), composed of two subunits, GapA and GapB (13, 14). GapB, up to now, has exclusively been found in Streptophyta, but recently two prasinophycean green algae, Ostreococcus tauri and Ostreococcus lucimarinus, were also shown to possess a GapB gene, whereas CP12 is missing (15). The GapB subunit is similar to the GapA subunit but has a C-terminal extension containing two redox-regulated cysteine residues (16). Thus, although the A₄ GAPDHs lack these regulatory cysteine residues (13, 14, 17–20), they are also redox-regulated through its interaction with CP12, since the C terminus of this small protein resembles the C-terminal extension of the GapB subunit. The regulatory cysteine residues for GapA are thus supplied by CP12, as is well documented in the literature (1, 8, 11, 16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs) (21–26). The amino acid composition of these proteins causes them to have no or few secondary structures. Their total or partial lack of structure and their high flexibility allow them to be molecular adaptors (27, 28). They are often able to bind to several partners and are involved in most cellular functions (29, 30). Recently, some IUPs have been described in photosynthetic organisms (31, 32).There are many functional categories of IUPs (22, 33). They can be, for instance, involved in permanent binding and have (i) a scavenger role, neutralizing or storing small ligands; (ii) an assembler role by forming complexes; and (iii) an effector role by modulating the activity of a partner molecule (33). These functions are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating its redox properties (8, 34, 35), and can also bind a metal ion (36, 37). IUPs can also bind transiently to partners, and some of them have been found to possess a chaperone activity (31, 38). This chaperone function was first shown for α-synuclein (39) and for α-casein (40), which are fully disordered. The amino acid composition of IUPs is less hydrophobic than those of soluble proteins; hence, they lack hydrophobic cores and do not become insoluble when heated. Since CP12 belongs to this family, we tested if it was resistant to heat treatment and finally, since it is tightly bound to GAPDH, if it could prevent aggregation of its partner, GAPDH, an enzyme well known for its tendency to aggregate (41–44) and consequently a substrate commonly used in chaperone studies (45, 46).Unlike chaperones, which form transient, dynamic complexes with their protein substrates through hydrophobic interactions (47, 48), CP12 forms a stable complex with GAPDH. The interaction involves the C-terminal part of the protein and the presence of negatively charged residues on CP12 (35). However, only a site-directed mutagenesis has been performed to characterize the interaction site on GAPDH. Although the mutation could have an indirect effect, the residue Arg-197 was shown to be a good candidate for the interaction site (49).In this report, we accordingly used proteolysis experiments coupled with mass spectrometry to detect which regions of GAPDH are protected by its association with CP12. To conclude, the aim of this report was to characterize a chaperone function of CP12 that had never been described before and to map the interaction site on GAPDH using an approach that does not involve site-directed mutagenesis.     

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [¹⁴C]gibberellin (GA)₉ and [¹⁴C]GA₂₀ to radiolabeled GA₄ and GA₁, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA₉ was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA₂₀. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA₁₂-aldehyde, GA₁₂, GA₁₉, GA₂₅, GA₅₃, or GA₄₄ as the open lactone (20-hydroxy-GA₅₃), whereas GA₁₅, GA₂₄, and GA₄₄ were hydroxylated to GA₃₇, GA₃₆, and GA₃₈, respectively. The open lactone of GA₁₅ (20-hydroxy-GA₁₂) was hydroxylated but less efficiently than GA₁₅. In contrast to the free acid, GA₂₅ 19,20-anhydride was 3β-hydroxylated to give GA₁₃. 2,3-Didehydro-GA₉ and GA₅ were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA₉ and GA₆.Dwarf mutants with reduced biosynthesis of the GA plant hormones have been valuable tools in studies of the function of these compounds (Ross, 1994). In Arabidopsis thaliana, mutations at six loci (GA1-GA6) that result in reduced GA biosynthesis have been identified (Koorneef and van der Veen, 1980; Sponsel et al., 1997), and three of these loci have recently been cloned. The GA1 locus was isolated by genomic subtraction (Sun et al., 1992) and shown by heterologous expression in Escherichia coli to encode the enzyme that cyclizes geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya, 1994). This enzyme was formerly referred to as ent-kaurene synthase A but has been renamed copalyl diphosphate synthase (Hedden and Kamiya, 1997; MacMillan, 1997). The GA5 locus was shown to correspond to one of the GA 20-oxidase genes (Xu et al., 1995), the products of which catalyze the conversion of GA₁₂ to GA₉ and GA₅₃ to GA₂₀ (Phillips et al., 1995; Xu et al., 1995). GA 20-oxidases are 2-oxoglutarate-dependent dioxygenases that are encoded by small multigene families, members of which are differentially expressed in plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the basis of the derived amino acid sequence, was also shown to encode a dioxygenase (Chiang et al., 1995). Several lines of evidence indicate that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a ga4 mutant, all alleles of which are semidwarf, contained reduced concentrations of the 3β-hydroxy GAs GA₁, GA₄, and GA₈ compared with the Landsberg erecta wild type, whereas levels of immediate precursors to these GAs were elevated (Talon et al., 1990). Furthermore, metabolism of [¹³C]GA₂₀ to [¹³C]GA₁ was substantially less in the mutant than in the wild type (Kobayashi et al., 1994). In the present paper we confirm by functional expression of its cDNA in E. coli that GA4 encodes a GA 3β-hydroxylase. In addition, we determine the substrate specificity of recombinant GA4 using a number of C₂₀- and C₁₉-GAs and show by kinetic analysis that the enzyme has a higher affinity for GA₉ than for GA₂₀, which is consistent with the non-13-hydroxylation pathway predominating in Arabidopsis (Talon et al., 1990).