MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells

MicroRNAs （or miRNAs） are small non-coding RNAs （21-25 nucleotides） that are involved in a wide range of activities related to the development and differentiation of cells. Comparison of the miRNA expression profiles of mouse P19 embryonic carcinoma cells with those of differentiated neural stem cells showed that the expression level of 65 miRNAs changed （2-fold） after differentiation. MiR-124a was dramatically upregulated （more than 20-fold） while miRNAs of the miR-302 family and those in the miR-290-295 cluster were strongly down-regulated. Further analysis revealed that some important factors such as Oct4 and Sox2 appeared to be involved in the regulation of these miRNAs. These results may contribute to a better understanding of miRNA-regulated neural differentiation in early mouse embryos.     

Targeted Gene Knockouts Reveal Overlapping Functions of the Five Physcomitrella patens FtsZ Isoforms in Chloroplast Division,Chloroplast Shaping,Cell Patterning,Plant Development,and Gravity Sensing

Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in R patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss R patens.     

Alpha-lipoic acid enhances DMSO-induced cardiomyogenic differentiation of P19 cells

Alpha-lipoic acid （oL-LA） is a potent antioxidant that acts as an essential cofactor in mitochondrial dehydrogenase reac- tions, α-LA has been shown to possess anti-inflammatory and cytoprotective properties, and is used to improve symptoms of diabetic neuropathy. However, the role of α-LA in stem cell differentiation and the underlying molecular mechanisms remain unknown. In the present study, we showed that α-LA significantly promoted dimethyl sulfoxide （DMSO）-induced cardiomyogenic differentiation of mouse embryonic carcin- oma P19 cells. α-LA dose dependently increased beating embryonic body （EB） percentages of DMSO-differentiated P19 cells. The expressions of cardiac specific genes TNNT2, Nkx2.5, GATA4, MEF2C, and MLC2V and cardiac isoform of troponin T （cTnT）-positively stained cell population were significantly up-regulated by the addition of α-LA. We also demonstrated that the differentiation time after EB formation was critical for α-LA to take effect. Interestingly, without DMSO treatment, α-LA did not stimulate the cardi- omyogenic differentiation of P19 cells. Further investigation indicated that collagen synthesis-enhancing activity, instead of the antioxidative property, plays a significant role in the cardiomyogenic differentiation-promoting function of α-LA. These findings highlight the potential use of α-LA for regen- erative therapies in heart diseases.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

The Earliest Normal Flower from Liaoning Province,China

The early evolution of angiosperms has been a focus of intensive research for more than a century. The Yixian Formation in western Liaoning yields one of the earliest angiosperm macrofioras. Despite multitudes of angiosperm fossils uncovered, including Archaefructus and Sinocarpus, no bona fide normal flower has been dated to 125 Ma （mega-annum） or older. Here we report Callianthus dilae gen. et sp. nov. from the Yixian Formation （Early Cretaceous） in western Liaoning, China as the earliest normal flower known to date. The flower demonstrates a typical floral organization, including tepals, androecium, and gynoecium. The tepals are spatulate with parallel veins. The stamens have a slender filament, a globular anther, bristles at the anther apex, and in situ round-triangular pollen grains. The gynoecium is composed of two stylate carpels enclosed in a fleshy envelope, and develops into a ＂hip＂ when mature. Since the well-accepted history of angiosperms is not much longer than 125 Ma, Callianthus together with Chaoyangia, Archaefructus and Sinocarpus from the Yixian Formation demonstrate a surprisingly high diversity of angiosperms, implying a history of angiosperms much longer than currently accepted.     

Protein-Repairing Methionine Sulfoxide Reductases in Photosynthetic Organisms： Gene Organization,Reduction Mechanisms,and Physiological Roles

Methionine oxidation to methionine sulfoxide （MetSO） is reversed by two types of methionine sulfoxide reducrases （MSRs）, A and B, specific to the S- and R-diastereomers of MetSO, respectively. MSR genes are found in most organisms from bacteria to human. In the current review, we first compare the organization of the MSR gene families in photosynthetic organisms from cyanobacteria to higher plants. The analysis reveals that MSRs constitute complex families in higher plants, bryophytes, and algae compared to cyanobacteria and all non-photosynthetic organisms. We also perform a classification, based on gene number and structure, position of redox-active cysteines and predicted sub-cellular localization. The various catalytic mechanisms and potential physiological electron donors involved in the regeneration of MSR activity are then de- scribed. Data available from higher plants reveal that MSRs fulfill an essential physiological function during environmental constraints through a role in protein repair and in protection against oxidative damage. Taking into consideration the ex- pression patterns of MSR genes in plants and the known roles of these genes in non-photosynthetic cells, other functions of MSRs are discussed during specific developmental stages and ageing in photosynthetic organisms.     

Expression,Imprinting, and Evolution of Rice Homologs of the Polycomb Group Genes

Polycomb group proteins （PcG） play important roles in epigenetic regulation of gene expression. Some core PeG proteins, such as Enhancer of Zeste （E（z））, Suppressor of Zeste （12） （Su（z）12）, and Extra Sex Combs （ESC）, are conserved in plants. The rice genome contains two E（z）-Iike genes, OsiEZ1 and OsCLF, two homologs of Su（z）12, OsEMF2a and OsEMF2b, and two ESC-like genes, OsFIE1 and OsFIE2. OsFIE1 is expressed only in endosperm; the maternal copy is expressed while the paternal copy is not active. Other rice PcG genes are expressed in a wide range of tissues and are not imprinted in the endosperm. The two E（z）-Iike genes appear to have duplicated before the separation of the dicots and monocots; the two homologs of Su（z）12 possibly duplicated during the evolution of the Gramineae and the two ESC- like genes are likely to have duplicated in the ancestor of the grasses. No homologs of the Arabidopsis seed-expressed PcG genes MEA and FIS2 were identified in the rice genome. We have isolated T-DNA insertion lines in the rice homologs of three PcG genes. There is no autonomous endosperm development in these T-DNA insertion lines. One line with a T-DNA insertion in OsEMF2b displays pleiotropic phenotypes including altered flowering time and abnormal flower organs, suggesting important roles in rice development for this gene.     

Oxygation Enhances Growth,Gas Exchange and Salt Tolerance of Vegetable Soybean and Cotton in a Saline Vertisol

Impacts of salinity become severe when the soil is deficient in oxygen. OxygaUon （using aerated water for subsurface drip irrigation of crop） could minimize the impact of salinity on plants under oxygen-limiting soil environments. Pot experiments were conducted to evaluate the effects of oxygation （12% air volume/volume of water） on vegetable soybean （moderately salt tolerant） and cotton （salt tolerant） in a salinized vertisol at 2, 8, 14, 20 dS/m ECe. In vegetable soybean, oxygation increased above ground biomass yield and water use efficiency （WUE） by 13% and 22%, respectively, compared with the control. Higher yield with oxygation was accompanied by greater plant height and stem diameter and reduced specific leaf area and leaf Na^＋ and CI^- concentrations. In cotton, oxygation increased lint yield and WUE by 18% and 16%, respectively, compared with the control, and was accompanied by greater canopy light interception, plant height and stem diameter. Oxygation also led to a greater rate of photosynthesis, higher relative water content in the leaf, reduced crop water stress index and lower leaf water potential. It did not, however, affect leaf Na^＋ or CI^- concentration. Oxygation invariably increased, whereas salinity reduced the K^＋： Na^＋ ratio in the leaves of both species. Oxygation improved yield and WUE performance of salt tolerant and moderately tolerant crops under saline soil environments, and this may have a significant impact for irrigated agriculture where saline soils pose constraints to crop production.     

Towards Characterization of the Chloroplast NAD（P）H Dehydrogenase Complex

The NAD（P）H dehydrogenase （NDH） complex in chloroplast thylakoid membranes functions in cyclic electron transfer, and in chlororespiration. NDH is composed of at least 15 subunits, including both chloroplast- and nuclear-encoded proteins. During the past few years, extensive proteomic and genetic research on the higher plant NDH complex has been carried out, resulting in identification of several novel nuclear-encoded subunits. In addition, a number of auxiliary proteins, which mainly regulate the expression of chloroplast-encoded ndh genes as well as the assembly and stabilization of the NDH complex, have been discovered and characterized. In the absence of detailed crystallographic data, the structure of the NDH complex has remained obscure, and therefore the role of several NDH-associated nuclear-encoded proteins either as auxiliary proteins or structural subunits remains uncertain. In this review, we summarize the current knowledge on the subunit composition and assembly process of the chloroplast NDH complex. In addition, a novel oligomeric structure of NDH, the PSI/NDH supercomplex, is discussed.     

GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 （gnl2-1） and gn12-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

Molecular cloning,expression, and anti-tumor activity of a novel serine protease from Arenicola cristata

Arenicola cristata, a marine annelid, is a wellknown and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease ofA. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA （cDNA） of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the pro posed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was 26 kDa after overexpression in E. coli. The recombinant pro tein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma （ESCC） in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activa tion of caspase9 as well as downregulation of Mcl1 and Bcl2. Taken together, our findings indicated that the recom binant serine protease ofA. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.     

LSCHL4 from Japonica Cultivar,Which Is Allelic to NAL 1, Increases Yield of Indica Super Rice 93-11

The basic premise of high yield in rice is to improve leaf photosynthetic efficiency and coordinate the sourcesink relationship in rice plants. Quantitative trait loci （QTLs） related to morphological traits and chlorophyll content of rice leaves were detected at the stages of heading to maturity, and a major QTL （qLSCHL4） related to flag leaf shape and chlorophyll content was detected at both stages in recombinant inbred lines constructed using the indica rice cultivar 93-11 and the japonica rice cultivar Nipponbare. Map-based cloning and expression analysis showed that LSCHL4 is allelic to NAL1, a gene previously reported in narrow leaf mutant of rice. Overexpression lines transformed with vector carrying LSCHL4 from Nipponbare and a near-isogenic line of 93-11 （NIL-9311） had significantly increased leaf chlorophyll content, enlarged flag leaf size, and improved panicle type. The average yield of NIL-9311 was 18.70% higher than that of 93-11. These results indicate that LSCHL4 had a pleiotropic function. Exploring and pyramiding more high-yield alleles resem- bling LSCHL4 for super rice breeding provides an effective way to achieve new breakthroughs in raising rice yield and generate new ideas for solving the problem of global food safety.