鼠伤寒沙门氏菌嘌呤生物合成调控研究：Ⅸ.PURbox的突变分析2

为研究１６ｂｐ ＰＵＲｂｏｓ中８个完全保守的碱基中的２个碱基与ｐｕｒＲ＾＋阻遏蛋白结合中的功能,对它们分别作了定点突变,使其分别从Ｃ,Ｇ突变为Ｇ,Ａ。凝胶阻滞实验结果表明,含上述保守碱基突变的ＰＵＲ ｂｏｘ均不能与ｐｕｒＲ＾＋阻遏蛋白结合。证明这２个保守碱基对维持ＰＵＲｂｏｘ的功能是必须的,其中任一改变都导致ＰＵＲｂｏｘ功能的丧失。     

鼠伤寒沙门氏菌嘌呤生物合成调控研究XI.PUR box的突变分析

为研究１６ｂｐ ＰＵＲ ｂｏｘ中除第３位的Ｇ与第１４位的Ｃ外,余下６个完全保守碱基的４个在与ＰｕｒＲ阻遏蛋白结合中的功能,对它们分别做了定点突变,使其分别从Ｃ、Ａ、Ａ和Ｔ突变为Ｇ、Ｇ、Ｇ和Ｃ。凝胶阻滞实验结果表明,含上述指定突变的：ＰＵＲｂｏｘ均不能与ＰｒｕＲ阻遏蛋白结合。由此证明,这４个保守碱基对维持ＰＵＲ ｂｏｘ的功能是必须的,其中任一改变都导致ＰＵＲｂｏｘ功能的夹失。     

鼠伤寒沙门氏菌嘌呤生物合成调控研究

为研究16bp PUR box中8个完全保守的碱基中的2个碱基在与purR\++阻遏蛋白结合中的功能,对它们分别作了定点突变,使其分别从C,G突变为G,A。凝胶阻滞实验结果表明,含上述保守碱基突变的PUR box均不能与purR\++阻遏蛋白结合。证明这2个保守碱基对维持PUR box的功能是必须的,其中任一改变都导致PUR box功能的丧失。     

鼠伤寒沙门氏菌嘌呤生物合成基因表达的调控研究：Ⅱ.O^c突变体的…

已有研究证明,编码阻遏蛋白的调节基因ｐｕｒＲ能调节嘌呤从头合成途径中除ｐｕｒＢ外所有结构基因的表达。但迄今还缺乏阻遏蛋白与这些基因的操作基因相结合的直接证据。本文报道以嘌呤结构基因ｐｕｒＤ和ｐｕｒＧ的ＭｕｄＪ插入物为出发株,在外加过量腺嘌呤核苷（２ｍｍｏｌ／Ｌ）的ＭａｃＣｏｎｋｅｙ平板上通过选择红色菌落分离Ｏ＾ｃ突变体的结果,从上述两株出发株分别获得了８株和９株独立的消阻遏突变体。共转导分析和顺反     

鼠伤寒沙门氏菌嘌呤生物合成的调控研究X.purR琥珀突变体(am)的分离和氨基酸取代的初步研究

以超阻遏突变体３—１８为出发株,采用以乳糖为唯一碳源的ＮＣＥ平板的方法分离到４３９ 株调节突变体。通过转导引入ｔＲＮＡ抑制基因从中检测到 １１株 ｐｕｒＲ（ａｍ）候选株。共转导分 析证明,这些突变株的琥珀浪突变均发生在ｐｕｒＲ上。用 ｓｕｐＤ． ｓｕｐＥ和 ｓｕｐＦ分别对上述各ａｍｂｅｒ 突变体作了氨基酸取代实验,初步结果表明：同一氨基酸对ｐｕｒＲ不同位点（ａｍ）的氨基酸取 代,对ＰｕｒＲ调节功能有不同程度的影响。不同氨基酸（３种）对ｐｕｒＲ同一位点（ａｍ）的氨基酸取 代,对其调节功能的影响也存在差异。     

乙肝病毒核衣壳启动子内三链DNA的形成

用电泳迁移分析方法研究了２１ｎｔ脱氧寡核苷酸Ｇ３ＴＧ２ＴＧＴ２Ｇ５ＴＧ２ＴＧＴ（ＣＰ１）与１２９ｂｐ的乙肝病毒（ＨＢＶ）核衣壳启动子（Ｃｐ）片段内一位点结合形成的三链ＤＮＡ的特异性及稳定性．在克隆有ＨＢＶ基因组的质粒ｐＣＰ１０的酶切产物中,ＣＰ１仅与含Ｃｐ的１２９ｂｐ片段结合．在２０ｍｍｏｌ／ＬＭｇ２＋溶液中其解离常数（Ｋｄ）为１．４×１０－７ｍｏｌ／Ｌ．不同离子稳定三链ＤＮＡ的效果依次为ｓｐ４＋（精胺）＞Ｍｇ２＋＞Ｚｎ２＋＞Ｎａ＋＞Ｋ＋,离子之间存在相互竞争作用．比ＣＰ１多一误配碱基的脱氧寡核苷酸Ｇ２ＴＧ２ＴＧＴＧ３ＴＧ２ＴＧ２ＴＧ２Ｔ（ＣＰ２）在２０ｍｍｏｌ／ＬＭｇ２＋溶液中与Ｃｐ结合的Ｋｄ值约为ＣＰ１的１／７,而在６０ｍｍｏｌ／ＬＫ＋或５ｍｍｏｌ／ＬＺｎ２＋溶液中检测不到它与Ｃｐ的结合,这进一步显示了三链ＤＮＡ形成的特异性．细胞的生理离子浓度被认为是：Ｓｐ４＋１ｍｍｏｌ／Ｌ,Ｍｇ２＋１０ｍｍｏｌ／Ｌ,Ｋ＋１４０ｍｍｏｌ／Ｌ,因此,ＣＰ１在细胞内将能特异地与Ｃｐ结合并具有较好的稳定性．     

酵母PHO2蛋白及其变异体与PHO5USA体外的相互作用

酵母ＰＨＯ２蛋白及其变异体与ＰＨＯ５ＵＳＡ体外的相互作用杨军,敖世洲（中国科学院上海生物化学研究所分子生物学国家重点实验室,２０００３１）关键词酵母;ＰＨＯ２;突变;ＤＮＡ结合ＰＨＯ２是酵母阻遏型酸性磷酸酯酶基因转录的正调控因子［１］,由５５９个氨基...     

鼠伤寒沙门氏菌嘌呤生物合成的调控研究X.purR琥珀突变体（am）的分离

以超阻遏突变体３－１８为出发株,采用以乳糖为唯一碳源的ＮＣＥ平板的方法分离到４３９株调节突变体。通过转导引入ｔＲＮＡ抑制基因从中检测到１１株ｐｕｒＲ（ａｍ）侯选株。共转导分析证明,这些突变株的琥珀突变均发生在ｐｕｒＲ上。用ｓｕｐＤ、ｓｕｐＥ和ｓｕｐＦ分别对上述各ａｍｂｅｒ突变体作了氨基酸取代实验,初步结果表明：同一氨基酸对ｐｕｒＲ不同位点（ａｍ）的氨基酸取代,对ＰｕｒＲ调节功能有不同程度的影响;     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

SAPGTP和PG为接头的5'IL6-TNFα衍生物融合蛋白

通过计算机模拟比较十种理论上柔性较好的接头在 ５′ Ｉ Ｌ６ Ｔ Ｎ ＦΔ融合蛋白中对 Ｉ Ｌ ６ 和 Ｔ Ｎ ＦΔ空间结构的影响情况,从中选择了 Ｓ Ａ Ｐ Ｇ Ｔ Ｐ接头．以 Ｓ Ａ Ｐ Ｇ Ｔ Ｐ 作为接头的 ５′ Ｉ Ｌ６ Ｓ Ａ Ｐ Ｇ Ｔ Ｐ Ｔ Ｎ ＦΔ和以 Ｐ Ｇ 为接头的５′ Ｉ Ｌ６ Ｐ Ｇ Ｔ Ｎ ＦΔ空间结构预测结果相似． Ｄ Ｎ Ａ 序列分析两种蛋白的接头序列均与设计的一致．５′ Ｉ Ｌ６ Ｓ Ａ Ｐ Ｇ Ｔ Ｐ Ｔ Ｎ ＦΔ和 ５′ Ｉ Ｌ６ Ｐ Ｇ Ｔ Ｎ ＦΔ蛋白的大肠杆菌表达产物经初步分离、纯化及鉴定后,生物学活性及对高表达 Ｉ Ｌ ６ 受体肿瘤细胞的杀伤作用比较结果显示：在 Ｌ９２９细胞上,前者的生物学活性是后者的 ２７ 倍;在 Ｕ９３７ 细胞上,前者对肿瘤细胞的抑制率是后者的１３ 倍．它们对高表达 Ｉ Ｌ ６ 受体的 Ｕ９３７ 细胞杀伤作用分别是同样突变位点的人 Ｔ Ｎ Ｆα衍生物的３７ 和 ２９ 倍．实验表明, Ｓ Ａ Ｐ Ｇ Ｔ Ｐ作为接头构建的 ５′ Ｉ Ｌ６ Ｓ Ａ Ｐ Ｇ Ｔ Ｐ Ｔ Ｎ ＦΔ融合蛋白优于以 Ｐ Ｇ 作为接头构建的 ５′ Ｉ Ｌ６ Ｐ Ｇ Ｔ Ｎ ＦΔ融合蛋白．     

Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities

The SCF ubiquitin ligase complex regulates diverse cellular functions by ubiquitinating numerous protein substrates. Cand1, a 120 kDa HEAT repeat protein, forms a tight complex with the Cul1-Roc1 SCF catalytic core, inhibiting the assembly of the multisubunit E3 complex. The crystal structure of the Cand1-Cul1-Roc1 complex shows that Cand1 adopts a highly sinuous superhelical structure, clamping around the elongated SCF scaffold protein Cul1. At one end, a Cand1 beta hairpin protrusion partially occupies the adaptor binding site on Cul1, inhibiting its interactions with the Skp1 adaptor and the substrate-recruiting F box protein subunits. At the other end, two Cand1 HEAT repeats pack against a conserved Cul1 surface cleft and bury a Cul1 lysine residue, whose modification by the ubiquitin-like protein, Nedd8, is able to block Cand1-Cul1 association. Together with biochemical evidence, these structural results elucidate the mechanisms by which Cand1 and Nedd8 regulate the assembly-disassembly cycles of SCF and other cullin-dependent E3 complexes.     

Recruitment of the inhibitor Cand1 to the cullin substrate adaptor site mediates interaction to the neddylation site

Cand1 inhibits cullin RING ubiquitin ligases by binding unneddylated cullins. The Cand1 N-terminus blocks the cullin neddylation site, whereas the C-terminus inhibits cullin adaptor interaction. These Cand1 binding sites can be separated into two functional polypeptides which bind sequentially. C-terminal Cand1 can directly bind to unneddylated cullins in the nucleus without blocking the neddylation site. The smaller N-terminal Cand1 cannot bind to the cullin neddylation region without C-terminal Cand1. The separation of a single cand1 into two independent genes represents the in vivo situation of the fungus Aspergillus nidulans, where C-terminal Cand1 recruits smaller N-terminal Cand1 in the cytoplasm. Either deletion results in an identical developmental and secondary metabolism phenotype in fungi, which resembles csn mutants deficient in the COP9 signalosome (CSN) deneddylase. We propose a two-step Cand1 binding to unneddylated cullins which initiates at the adaptor binding site and subsequently blocks the neddylation site after CSN has left.     

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8^AN015 or CSN5^null mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.     

Two G-protein-coupled-receptor candidates, Cand2 and Cand7, are involved in Arabidopsis root growth mediated by the bacterial quorum-sensing signals N-acyl-homoserine lactones

Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules to coordinate their group behavior. Recently, it was shown that plants can perceive and respond to these bacterial AHLs. However, little is known about the molecular mechanism underlying the response of plants to bacterial QS signals. In this study, we show that the promotion of root elongation in wild type Arabidopsis thaliana induced by the AHLs N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) or N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) was completely abolished in plants with loss-of-function mutations in two candidate G-protein Coupled Receptors (GPCRs), Cand2 and Cand7. Furthermore, real-time PCR analysis revealed that the expression levels of Cand2 and Cand7 were elevated in plants treated with 3OC6-HSL or 3OC8-HSL. These results suggest that Cand2 and Cand7 are involved in the regulation of root growth by bacterial AHLs and that GPCRs play a role in mediating interactions between plants and microbes.     

Cullin neddylation and substrate‐adaptors counteract SCF inhibition by the CAND1‐like protein Lag2 in Saccharomyces cerevisiae

Cullin‐based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin‐like protein Nedd8. Cullin neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non‐neddylated yeast cullin Cdc53 and prevents its neddylation in vivo and in vitro. Binding occurs through a conserved C‐terminal β‐hairpin structure that inserts into the Skp1‐binding pocket on the cullin, and an N‐terminal motif that covers the neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N‐terminal‐binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate‐specific adaptors triggers release of Cand1/Lag2, whereas subsequent neddylation of the cullin facilitates the removal and prevents re‐association of Lag2/Cand1.     

A note on the yeast flora associated with fermentation of mango

Fifteen morphologically different groups of yeasts consisting of Metschnikowia pulcherrima, Trichosporon cutaneum, Kloeckera apiculata, Torulopsis Candida, Tor. glabrata. Tor. apicola, Candida tropicalis, Cand. krusei, Cand. sorbosa, Cand. diversa, Pichia terricola, Pic. membranaefaciens, Hyphopichia burtonii, Rhodotorula graminis and Aureobasidium pullulans were isolated from fresh, fermenting and fermented juice of two varieties of mango. This is the first report on the study of yeast flora of mango and also on the occurrence of Hyp. burtonii on fruits.     

Set them free: F-box protein exchange by Cand1

Cand1 (Cullin-associated and neddylation-dissociated protein 1) has long been known as a regulator of SCF ubiquitin ligases, but details remained puzzling due to conflicting results from in vitro and in vivo experiments. Three recent reports, one in Cell and two in Nature Communications, propose Cand1 as a protein exchange factor with interesting mechanism that reconciles Cand1 genetics and biochemistry.Most eukaryotic proteins are modified by the small protein ubiquitin at some point during their life. Ubiquitin tags can mark them for degradation in the proteasome, or control other protein properties such as localization, activity, and interactions. Ubiquitin ligases (E3 enzymes) play a particularly important role in the E1-E2-E3 ubiquitylation cascade as they directly select substrates for ubiquitin attachment. E3s define a large protein family with over 600 members in human cells that control ubiquitin transfer onto thousands of substrate proteins¹. The complexity of this system comes with conceptual challenges that are particularly apparent for the largest group of E3s, the multisubunit Cullin-RING ubiquitin ligases (CRLs). The archetypal CRLs, the Skp1/Cul1/F-box protein (SCF) complexes, assemble on the Cul1 scaffold, with the small RING protein Rbx1 and E2 bound to the C-terminus, and the adapter protein Skp1 associated with the N-terminal region. Skp1 binds to one of many F-box proteins (FboxP), which confer specificity by selectively recruiting substrate proteins for ubiquitin transfer² (Figure 1, right). Up to 69 FboxPs in humans, and possibly 700 in plants, compete for the Cul1 core. How cells adjust abundance of the different SCF ligases in response to cell cycle and environmental cues to dynamically match substrate demand is one of the major questions in the field. Since identification of Cand1 over 10 years ago, its involvement in SCF complex formation has been evident^3,4. However, its true function was somewhat of a mystery. Cand1 acted as a potent SCF inhibitor in vitro by displacing the FboxP-Skp1 pair from Cul1, but genetic experiments classified Cand1 as a positive regulator of SCF and other CRLs in vivo⁵. An additional layer of complexity is added by covalent modification of cullins with the ubiquitin-like protein Nedd8. Neddylation (modification with Nedd8) induces a conformational rearrangement of Cul1 that stimulates ubiquitin transfer by the SCF-bound E2 and also obscures the Cand1 binding site on Cul1⁶. Nedd8 deconjugation is catalyzed by the COP9 signalosome (CSN). Strikingly, the paradox observed for Cand1 is also evident for CSN, because CSN clearly functions as a negative regulator of SCF in vitro, yet genetic data suggest a positive role for SCF activity in vivo⁵. A prevailing model has been that SCF and other CRLs must undergo neddylation cycles whereby deneddylated cullins are sequestered by Cand1, allowing substrate receptor exchange followed by reactivation of the assembled CRL by neddylation. However, mechanistic insight was scarce.Open in a separate windowFigure 1Cand1-driven substrate receptor exchange model (based on Pierce et al.⁷). Substrate availability protects the stable substrate ubiquitylation state (right). Depletion of substrates enhances CSN-mediated deneddylation shifting the SCF complex into a transition state that either finds new substrates and becomes reactivated by Nedd8 (N8) conjugation, or forms a transient complex with Cand1. The transient complex is highly unstable because of steric interference between F-box protein and Cand1 causing cycles of Cand1 and FboxP-Skp1 eviction. The exchange state allows the repertoire of formed SCF complexes to sample for substrates and, upon engagement, transit into the stable substrate ubiquitylation state.In a recent study published in Cell⁷, Deshaies and colleagues provide a biochemical framework that not only explains the CSN and Cand1 paradoxes, but also suggests a model for how SCF composition adjusts to varying substrate demand. They used in vitro real-time fluorescence resonance energy transfer (FRET) assays to monitor binding dynamics between FboxP-Skp1 and Cul1-Rbx1 complexes. Fbxw7-Skp1 formed an astonishingly tight complex with Cul1-Rbx1 (K_D = 200 fM) that could not be replaced by other FboxP-Skp1 complexes. However, addition of Cand1 accelerated spontaneous dissociation of SCF^Fbxw7 over one-million-fold. Kinetic measurements demonstrated that Cand1 acts neither as a competitive nor allosteric inhibitor of Fbxw7-Skp1 binding to Cul1-Rbx1. Instead, Cand1 specifically increases the dissociation rate of the FboxP-Skp1 complex while having little effect on association rates. The authors point out that such a kinetic effect is reminiscent of guanine nucleotide exchange factors (GEFs). Accordingly, they suggest the term substrate receptor exchange factor (SREF) for Cand1 and functionally similar factors.Cand1''s SREF activity was beautifully illustrated in vitro using the two different F-box proteins Fbxw7 and β-TrCP. When SCF^β-TrCP was combined with purified Fbxw7-Skp1 in an in vitro ubiquitylation reaction, no ubiquitylation of cyclin E (Fbxw7 substrate) was observed. This was not surprising because the tight binding of β-TrCP-Skp1 to Cul1 was expected to prevent assembly of SCF^Fbxw7. Remarkably, addition of Cand1 dramatically stimulated cyclin E ubiquitylation, likely through dissociation of β-TrCP-Skp1, thus establishing a new equilibrium of SCF^β-TrCP and SCF^Fbxw7 complexes. This assay design exposed Cand1 as an activator of SCF in vitro, which is consistent with its positive regulator role revealed by genetic experiments. The important findings that the FboxP-Skp1 complex can remove tightly bound Cand1 from Cul1, and indication of a transient complex of Cand1 with fully assembled SCF led to proposal of a model for SCF dynamics driven by substrate demand (Figure 1). A key feature of the model is based on recent evidence that substrate binding to CRLs can significantly reduce CSN access and CRL deneddylation^8,9. When substrates are exhausted, accelerated deneddylation shifts the active SCF complex into a deneddylated transition state, which can either bind new substrate and become reactivated by Nedd8 conjugation, or enter the exchange state. The latter is characterized by a Cand1-bound transition complex that controls dissociation and association of FboxP-Skp1 complexes. This concept extends the previous neddylation cycle model based on a strong biochemical foundation and provides a hypothesis for dynamic remodeling of the SCF landscape by substrate demand. Pierce et al.⁷ support this biochemical concept with findings in vivo demonstrating significant shifts in the SCF landscape when Cand1 is absent.The importance of Cand1 as a F-box protein exchange factor is reinforced by two recent studies in yeast. Zelma et al.¹⁰ demonstrate the role of Cand1 in remodeling the SCF repertoire in response to changing growth conditions, and Wu et al.¹¹ provide additional evidence for Cand1 as an F-box protein exchange factor in vivo. Clearly there are more challenges ahead to understanding CRL dynamics, but the significance of these findings may reach beyond ubiquitin biology as it introduces the concept of protein exchange factors that govern association of protein binding platforms with large numbers of interactors.     

Insights into the metabolism,lifestyle and putative evolutionary history of the novel archaeal phylum ‘Diapherotrites'

The archaeal phylum ‘Diapherotrites'' was recently proposed based on phylogenomic analysis of genomes recovered from an underground water seep in an abandoned gold mine (Homestake mine in Lead, SD, USA). Here we present a detailed analysis of the metabolic capabilities and genomic features of three single amplified genomes (SAGs) belonging to the ‘Diapherotrites''. The most complete of the SAGs, Candidatus ‘Iainarchaeum andersonii'' (Cand. IA), had a small genome (∼1.24 Mb), short average gene length (822 bp), one ribosomal RNA operon, high coding density (∼90.4%), high percentage of overlapping genes (27.6%) and low incidence of gene duplication (2.16%). Cand. IA genome possesses limited catabolic capacities that, nevertheless, could theoretically support a free-living lifestyle by channeling a narrow range of substrates such as ribose, polyhydroxybutyrate and several amino acids to acetyl-coenzyme A. On the other hand, Cand. IA possesses relatively well-developed anabolic capabilities, although it remains auxotrophic for several amino acids and cofactors. Phylogenetic analysis suggests that the majority of Cand. IA anabolic genes were acquired from bacterial donors via horizontal gene transfer. We thus propose that members of the ‘Diapherotrites'' have evolved from an obligate symbiotic ancestor by acquiring anabolic genes from bacteria that enabled independent biosynthesis of biological molecules previously acquired from symbiotic hosts. ‘Diapherotrites'' 16S rRNA genes exhibit multiple mismatches with the majority of archaeal 16S rRNA primers, a fact that could be responsible for their observed rarity in amplicon-generated data sets. The limited substrate range, complex growth requirements and slow growth rate predicted could be responsible for its refraction to isolation.     

The Effects of Temperature on Action Potentials in the Chill Sensitive Seismonastic Plant Biophytum sensitivum

The seismonastic plant Biophytum sensitivum was stimulated electricallyto produce reproducible action potentials. It was found thatnegative voltages only induced a response and that stimulationcould also be achieved by electrostatic means by depositingnegative charge on the leaf surface. A study of the effectsof temperature on the propagation rates of action potentialsin different aged leaves showed that for leaves of all agesthe fastest propagation occurred at 24 ?C. As the temperaturewas decreased or increased from 24 ?C a reduction in the propagationrate occurred. No propagation was found at, or below, 14 ?Cand the propagation rate decreased with increasing leaf age.Both the threshold voltage level and the charge required toproduce action potentials in young leaves was lowest at 24 ?Cand had to be increased at either lower or higher temperaturesto cause leaf closure. The duration of the electrical stimulusrequired to induce action potentials was found to decrease asthe voltage was increased. Mechanisms which could account forthe effects of temperature on action potentials are discussedin relation to the chill-sensitivity of the plant. Key words: Chilling, Action Potentials, Biophytum sensitivum     

Effects of ANG II type 1 and 2 receptors on oxidative stress,renal NADPH oxidase,and SOD expression

Oxidative stress accompanies angiotensin (ANG) II infusion, but the role of ANG type 1 vs. type 2 receptors (AT1-R and AT2-R, respectively) is unknown. We infused ANG II subcutaneously in rats for 1 wk. Excretion of 8-isoprostaglandin F2alpha (8-Iso) and malonyldialdehyde (MDA) were related to renal cortical mRNA abundance for subunits of NADPH oxidase and superoxide dismutases (SODs) using real-time PCR. Subsets of ANG II-infused rats were given the AT1-R antagonist candesartan cilexetil (Cand) or the AT2-R antagonist PD-123,319 (PD). Compared to vehicle (Veh), ANG II increased 8-Iso excretion by 41% (Veh, 5.4 +/- 0.8 vs. ANG II, 7.6 +/- 0.5 pg/24 h; P < 0.05). This was prevented by Cand (5.6 +/- 0.5 pg/24 h; P < 0.05) and increased by PD (15.8 +/- 2.0 pg/24 h; P < 0.005). There were similar changes in MDA excretion. Compared to Veh, ANG II significantly (P < 0.005) increased the renal cortical mRNA expression of p22phox (twofold), Nox-1 (2.6-fold), and Mn-SOD (1.5-fold) and decreased expression of Nox-4 (2.1-fold) and extracellular (EC)-SOD (2.1-fold). Cand prevented all of these changes except for the increase in Mn-SOD. PD accentuated changes in p22phox and Nox-1 and increased p67phox. We conclude that ANG II infusion stimulates oxidative stress via AT1-R, which increases the renal cortical mRNA expression of p22phox and Nox-1 and reduces abundance of Nox-4 and EC-SOD. This is offset by strong protective effects of AT2-R, which are accompanied by decreased expression of p22phox, Nox-1, and p67phox.