Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

An active CO₂-concentrating mechanism is induced when Chlamydomonas reinhardtii acclimates to limiting inorganic carbon (Ci), either low-CO₂ (L-CO₂; air level; approximately 0.04% CO₂) or very low-CO₂ (VL-CO₂; approximately 0.01% CO₂) conditions. A mutant, ad1, which is defective in the limiting-CO₂-inducible, plastid-localized LCIB, can grow in high-CO₂ or VL-CO₂ conditions but dies in L-CO₂, indicating a deficiency in a L-CO₂-specific Ci uptake and accumulation system. In this study, we identified two ad1 suppressors that can grow in L-CO₂ but die in VL-CO₂. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of L-CO₂-acclimated suppressors under acclimation CO₂ concentrations were more than 2-fold higher than ad1, apparently resulting from a more than 20-fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. However, photosynthetic rates of VL-CO₂-acclimated cells under acclimation CO₂ concentrations were too low to support growth in spite of a significantly elevated intracellular Ci concentration. We conclude that LCIB functions downstream of CAH3 in the CO₂-concentrating mechanism and probably plays a role in trapping CO₂ released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO₂ to support growth in L-CO₂-acclimated cells, but not in VL-CO₂-acclimated cells, even in the absence of LCIB.CO₂ serves both as the substrate for photosynthesis and as an important signal to regulate plant growth and development, so variable CO₂ concentrations can impact photosynthesis, growth, and productivity of plants. Terrestrial C₄ plants have developed a CO₂-concentrating mechanism (CCM) involving anatomical and biochemical adaptations to accumulate a higher concentration of CO₂ as substrate Rubisco and to suppress oxygenation of ribulose-1,5-bisP, a wasteful side reaction. In contrast, a different type of CCM is induced in the unicellular green microalga Chlamydomonas reinhardtii when the supply of dissolved inorganic carbon (Ci; CO₂ and HCO₃⁻) for photosynthesis is limited (Beardall and Giordano, 2002; Giordano et al., 2005; Moroney and Ynalvez, 2007; Spalding, 2008). In response to limiting CO₂, the CCM uses active Ci transport, both at the plasma membrane and the chloroplast envelope, to accumulate a high concentration of HCO₃⁻ within the chloroplast (Palmqvist et al., 1988; Sültemeyer et al., 1988). The thylakoid lumen carbonic anhydrase (CAH3) plays an essential role in the rapid dehydration of the accumulated HCO₃⁻ to release CO₂ into the pyrenoid, a Rubisco-containing internal compartment of the chloroplast, for assimilation by Rubisco (Price et al., 2002; Spalding et al., 2002).While a number of genes and proteins essential to the operation of the CCM in C. reinhardtii have been identified, our understanding of Ci uptake and its regulation, as well as other aspects of CCM function is limited. A better understanding of the similar CCM in prokaryotic organisms, specifically the cyanobacteria Synechocystis and Synechococcus, has been gained. At least five different types of Ci transporters have been identified in cyanobacteria, including three HCO₃⁻ transporters and two active CO₂ uptake systems (Price et al., 2002, 2004).Recently, at least three distinct CO₂-regulated acclimation states were identified in C. reinhardtii based on growth, photosynthesis and gene expression characteristics, a high-CO₂ (H-CO₂) state (5%–0.5% CO₂), low-CO₂ (L-CO₂) state (air level; 0.4%–0.03% CO₂), and very low-CO₂ (VL-CO₂) state (0.01%–0.005% CO₂; Vance and Spalding, 2005). Two allelic HCR (H-CO₂-requiring) mutants, pmp1 and ad1, grow as well (pmp1) or nearly as well (ad1) as wild-type cells in both H-CO₂ and VL-CO₂ conditions while only dying in L-CO₂, indicating a deficient Ci transport and/or accumulation system only in the L-CO₂ acclimation state (Spalding et al., 1983b, 2002). The defective gene responsible for the pmp1/ad1 phenotype was identified as LCIB, a limiting CO₂-inducible gene, the product of which is predicted to be located in the chloroplast stroma and proposed to be involved with chloroplast Ci uptake in L-CO₂ conditions (Wang and Spalding, 2006). The LCIB gene product is a member of a small gene family so far only found in a few microalgae species (Spalding, 2008).To investigate the roles of LCIB in eukaryotic photosynthetic organisms and identify other functional components involved in chloroplast Ci accumulation in C. reinhardtii, we used an insertional mutagenesis approach to select suppressors of the air-dier phenotype of the LCIB mutant ad1. In this study, we describe two ad1 suppressors, ad-su6 and ad-su7, that grow normally in L-CO₂ but, unlike ad1, die in VL-CO₂. This report also presents data suggesting that the air-dier phenotype of ad1 is suppressed by increased intracellular Ci concentrations in the two suppressors, and suggesting a possible role for LCIB as a CO₂ trap rather than having any direct role in chloroplast envelope Ci transport.     

四株盐杆菌中类紫质分子的探测

研究了四株盐杆菌Halobacterium sp. xiz 515.H.sp.dal s_2-1和H.sp.ausH_3a光驱动的质子泵功能及相应视黄醛蛋白的光化学反应.用pH电极测定了细胞悬浮液和胞外被膜(cellenvelope)介质中的光致△pH变化.实验发现,xiz.515和dals_2-1在照光时介质酸化.而另两株则相反,介质碱化.在这些盐杆菌中,进行了细胞膜各组份离心分离,其操作过程如同在Hhalobium上R中分离紫膜一样.最后得到的相应于紫膜组份的细胞膜中,没有发现表微菌紫质(BR)分子存在的吸收峰值.但是,在动力学光谱仪上却都发现此组份内具有与BR分子类似的光循环反应.本文假定,在这些盐杆菌中存在有微量的BR或类BR分子,它们以单体形式分散的小的聚集体散落在部份细胞膜上.     

Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

Extremely Low Frequency Magnetic Fields (ELF MF) has been considered as a “possible human carcinogen” by International Agency for Research on Cancer (IARC) while credible mechanisms of its carcinogenicity remain unknown. In this study, a proteomics approach was employed to investigate the changes of protein expression profile induced by ELF MF in human breast cancer cell line MCF7, in order to determine ELF MF-responsive proteins. MCF7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h and the changes of protein profile were examined using two dimensional electrophoresis. Up to 6 spots have been statistically significantly altered (their expression levels were changed at least 5 fold up or down) compared with sham-exposed group. 19 ones were only detected in exposure group while 19 ones were missing. Three proteins were identified by LC-IT Tandem MS as RNA binding protein regulatory subunit Proteasome subunit beta type 7 precursor and Translationally Controlled Tumor Protein. Our finding showed that 50 Hz, 0.4 mT ELF MF alternates the protein profile of MCF7 cell and may affect many physiological functions of normal cell and 2-DE coupled with MS is a promising approach to elucidating cellular effects of electromagnetic fields.     

Manganese ethylene-bis-dithiocarbamate and selective dopaminergic neurodegeneration in rat: a link through mitochondrial dysfunction

Manganese ethylene-bis-dithiocarbamate (Mn-EBDC) is the major active element of maneb, a pesticide linked to parkinsonism in certain individuals upon chronic exposure. Additionally, it has been shown to produce dopaminergic neurodegeneration in mice systemically coexposed to another pesticide, 1,1'-dimethyl-4,4'-bipyridinium (paraquat). Here, we described a rat model in which selective dopaminergic neurodegeneration was produced by delivering Mn-EBDC directly to the lateral ventricles. After establishing this model, we tested whether Mn-EBDC provoked dopamine efflux in the striatum, a well-known phenomenon produced by the mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes parkinsonism in humans, as well as in some animals. Finally, we investigated whether Mn-EBDC directly inhibited mitochondrial function in vitro using isolated brain mitochondria. Our data demonstrated that Mn-EBDC induced extensive striatal dopamine efflux that was comparable with that induced by MPP+, and that Mn-EBDC preferentially inhibited mitochondrial complex III. As mitochondrial dysfunction is pivotal in the pathogenesis of Parkinson's disease (PD), our results support the proposal that exposure to pesticides such as maneb, or other naturally occurring compounds that inhibit mitochondrial function, may contribute to PD development.     

Expansion of bilin-based red light sensors in the subaerial desert cyanobacterium Nostoc flagelliforme

Light is the crucial environmental signal for desiccation-tolerant cyanobacteria to activate photosynthesis and prepare for desiccation at dawn. However, the photobiological characteristics of desert cyanobacteria adaptation to one of the harshest habitats on Earth remain unresolved. In this study, we surveyed the genome of a subaerial desert cyanobacterium Nostoc flagelliforme and identified two phytochromes and seven cyanobacteriochromes (CBCRs) with one or more bilin-binding GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains. Biochemical and spectroscopic analyses of 69 purified GAF-containing proteins from recombinant phycocyanobilin (PCB), biliverdin or phycoerythrobilin-producing Escherichia coli indicated that nine of these proteins bind chromophores. Further investigation revealed that 11 GAFs form covalent adducts responsive to near-UV and visible light: eight GAFs contained PCB chromophores, three GAFs contained biliverdin chromophores and one contained the PCB isomer, phycoviolobilin. Interestingly, COO91_03972 is the first-ever reported GAF-only CBCR capable of sensing five wavelengths of light. Bioinformatics and biochemical analyses revealed that residue P132 of COO91_03972 is essential for chromophore binding to dual-cysteine CBCRs. Furthermore, the complement of N. flagelliforme CBCRs is enriched in red light sensors. We hypothesize that these sensors are critical for the acclimatization of N. flagelliforme to weak light environments at dawn.     

Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology

Production of therapeutic monoclonal antibodies using genetically modified plants may provide low cost, high scalability and product safety; however, antibody purification from plants presents a challenge due to the large quantities of biomass that need to be processed. Protein A column chromatography is widely used in the pharmaceutical industry for antibody purification, but its application is limited by cost, scalability and column fouling problems when purifying plant-derived antibodies. Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds, are relatively inexpensive to produce and provide a new approach for the capture of monoclonal antibodies from plants. When Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase while impurities remain in the aqueous phase. This is followed by repeated partitioning of oilbody phase against an aqueous phase via centrifugation to remove impurities before purified antibody is eluted from the oilbodies. We have developed this purification process to recover trastuzumab, an anti-HER2 monoclonal antibody used for therapy against specific breast-cancers that over express HER2 (human epidermal growth factor receptor 2), from transiently infected Nicotiana benthamiana. Protein A-OB overcomes the fouling problem associated with traditional Protein A chromatography, allowing for the development of an inexpensive, scalable and novel high-resolution method for the capture of antibodies based on simple mixing and phase separation.     

The actin multigene family in Populus: organization, expression and phylogenetic analysis

Despite the significance of actin in plant growth and development, little is known of the structure, expression and evolution of the actin gene family in woody plants. In this study, we systematically examined the diversification of the actin gene family in Populus by integrating genomic organization, expression, and phylogeny data. Genome-wide analysis of the Populus genome indicated that actin is a multigene family consisting of eight members, all predicted to encode 377-amino acid polypeptides that share high sequence homology ranging from 94.2 to 100% identity. Microarray and real-time PCR expression analysis showed that the PtrACT family members are differentially expressed in different tissues, exhibiting overlapping and unique expression patterns. Of particular interest, all PtrACT genes have been found to be preferentially expressed in the stem phloem and xylem, suggesting that poplar PtrACTs are involved in the wood formation. Gene structural and phylogenetic analyses revealed that the PtrACT family is composed of two main subgroups that share an ancient common ancestor. Extremely high intraspecies synonymous nucleotide diversity of π_syn = 0.01205 was detected, and the π_non-syn/π_syn ratio was significantly less than 1; therefore, the PtACT1 appears to be evolving in Populus, primarily under purifying selection. We demonstrated that the actin gene family in Populus is divided into two distinct subgroups, suggesting functional divergence. The results reported here will be useful in conducting future functional genomics studies to understand the detailed function of actin genes in tree growth and development.     

羽衣甘蓝BoMAPK4原核表达、抗体制备及蛋白表达分析

以羽衣甘蓝（Brassica oleracea var. acephala）自交不亲和系（S_13-bS_13-b ）为材料,提取柱头RNA,利用RT-PCR分离羽衣甘蓝柱头丝裂原活化蛋白激酶4（MAPK4）基因。结果表明：获得的BoMAPK4 cDNA含有一个 1 122 bp开放阅读框,编码373个氨基酸,具有丝氨酸/苏氨酸结构域,无信号肽和跨膜结构。羽衣甘蓝BoMAPK4与油菜（B. napus）BnMAPK4、芜菁（B. rapa）BrMAPK4、拟南芥（Arabidopsis thaliana）AtMAPK4的氨基酸序列一致性分别为99.7%、99.5%、95.4%。将BoMAPK4编码区的序列构建到原核表达载体pET-14b上,并转化到BL21（DE3）大肠杆菌（Escherichia coli）中进行原核表达。SDS-PAGE结果显示,在分子量大小约45 kDa处有BoMAPK4重组蛋白特异性表达。利用亲和层析的方法获得高纯度的重组BoMAPK4蛋白。利用获得的重组BoMAPK4蛋白免疫小鼠,制备BoMAPK4的多克隆抗体。提取羽衣甘蓝萼片、花瓣、花药、柱头、花柱和子房的总蛋白,利用免疫印迹的方法进行蛋白表达检测,结果发现BoMAPK4在花瓣、花药和子房中表达的较少,在萼片和花柱中表达的较多,在柱头中的表达量最高。这些研究为探讨BoMAPK4的生物学功能奠定了基础。     

乳酸链球菌素的分子结构、抗菌活性及基因工程研究

乳酸链球菌素(Nisin)是一种由34个氨基酸组成的天然抗菌素,对革兰氏阳性菌具有广谱抑菌作用。Nisin通过吸附于细胞膜,在膜上形成孔洞杀菌。Nisin的11个基因形成基因簇,负责其翻译后修饰、转运,先导序列的切除,成熟分子的外泌,合成过程的调节等。Nisin高产菌株的选育主要通过物理化学诱变,基因工程高效表达技术的研究刚刚开始。Nisin在食品和医药领域具有广泛的应用。本文主要就Nisin的分子结构、分泌机制、抗菌活性、作用机理,特别是它的基因工程技术研究进展等作一概述。     

MOABS: model based analysis of bisulfite sequencing data

Background

Human aging is associated with DNA methylation changes at specific sites in the genome. These epigenetic modifications may be used to track donor age for forensic analysis or to estimate biological age.

Results

We perform a comprehensive analysis of methylation profiles to narrow down 102 age-related CpG sites in blood. We demonstrate that most of these age-associated methylation changes are reversed in induced pluripotent stem cells (iPSCs). Methylation levels at three age-related CpGs - located in the genes ITGA2B, ASPA and PDE4C - were subsequently analyzed by bisulfite pyrosequencing of 151 blood samples. This epigenetic aging signature facilitates age predictions with a mean absolute deviation from chronological age of less than 5 years. This precision is higher than age predictions based on telomere length. Variation of age predictions correlates moderately with clinical and lifestyle parameters supporting the notion that age-associated methylation changes are associated more with biological age than with chronological age. Furthermore, patients with acquired aplastic anemia or dyskeratosis congenita - two diseases associated with progressive bone marrow failure and severe telomere attrition - are predicted to be prematurely aged.

Conclusions

Our epigenetic aging signature provides a simple biomarker to estimate the state of aging in blood. Age-associated DNA methylation changes are counteracted in iPSCs. On the other hand, over-estimation of chronological age in bone marrow failure syndromes is indicative for exhaustion of the hematopoietic cell pool. Thus, epigenetic changes upon aging seem to reflect biological aging of blood.