拟南芥中两个可能的SDD1新等位基因的鉴定与分析

SDD1是气孔发育过程中的关键调控基因,编码一个类枯草杆菌（Bacillus subtilis）蛋白酶的丝氨酸蛋白酶。从EMS诱变的拟南芥（Arabidopsis thaliana）中筛选到2株类似sdd1-1的气孔密度突变体,即e281和g204。其气孔密度和指数均比野生型增加约1.5倍,气孔成簇。遗传分析和基因测序证实它们是2个不同的SDD1新等位基因,其突变分别导致了底物结合位点N区域和催化三联体之一--S区域的氨基酸变化,分别为S变成T及S变为F。形态学和生理学研究表明,SDD1基因不同位点发生突变可导致不同的生物学效应;而且SDD1等位基因间存在拮抗作用,其可能属于基因转应作用中的负效应。     

拟南芥中两个可能的SDD1新等位基因的鉴定与分析

SDD1是气孔发育过程中的关键调控基因, 编码一个类枯草杆菌(Bacillus subtilis)蛋白酶的丝氨酸蛋白酶。从EMS诱变的拟南芥(Arabidopsis thaliana)中筛选到2株类似sdd1-1的气孔密度突变体, 即e281和g204。其气孔密度和指数均比野生型增加约1.5倍, 气孔成簇。遗传分析和基因测序证实它们是2个不同的SDD1新等位基因, 其突变分别导致了底物结合位点N区域和催化三联体之一——S区域的氨基酸变化, 分别为S变成T及S变为F。形态学和生理学研究表明, SDD1基因不同位点发生突变可导致不同的生物学效应; 而且SDD1等位基因间存在拮抗作用, 其可能属于基因转应作用中的负效应。     

Identification and Characterization of Circular RNAs As a New Class of Putative Biomarkers in Human Blood

Covalently closed circular RNA molecules (circRNAs) have recently emerged as a class of RNA isoforms with widespread and tissue specific expression across animals, oftentimes independent of the corresponding linear mRNAs. circRNAs are remarkably stable and sometimes highly expressed molecules. Here, we sequenced RNA in human peripheral whole blood to determine the potential of circRNAs as biomarkers in an easily accessible body fluid. We report the reproducible detection of thousands of circRNAs. Importantly, we observed that hundreds of circRNAs are much higher expressed than corresponding linear mRNAs. Thus, circRNA expression in human blood reveals and quantifies the activity of hundreds of coding genes not accessible by classical mRNA specific assays. Our findings suggest that circRNAs could be used as biomarker molecules in standard clinical blood samples.     

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.     

Identification and Characterization of Proteins Involved in Rice Urea and Arginine Catabolism

Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K_m = 67 mm, k_cat = 490 s⁻¹). The activity depended on the presence of manganese (K_d = 1.3 μm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.Nitrogen (N) availability often limits plant performance in natural ecosystems (Vitousek and Howarth, 1991), causing a selective pressure to optimize the use of N resources. This ecophysiological selection has even led to a reduction of the N content of plant proteins in comparison with animal orthologs (Elser et al., 2006). Because N is a limiting resource, plants do not only require efficient N uptake mechanisms but also possess enzymatic pathways for N remobilization.Arg is the most important single metabolite for N storage in plant seeds. In a survey of 379 plant species, Arg N accounted on average for 17.3% of total seed N (Vanetten et al., 1967). In several rice (Oryza sativa) varieties, values ranging from 16.1% to 17.1% were measured (Mosse et al., 1988). To access the N stored in the guanidinium group of Arg, it must first be hydrolyzed by mitochondrial arginase to Orn and urea. Urea leaves the mitochondria and is hydrolyzed by urease in the cytosol, releasing ammonia, which is reassimilated into amino acids by the combined action of Gln synthetase and Glu synthase.Urea not only originates from Arg breakdown but may also be taken up from the environment by urea transporters (Kojima et al., 2007; Wang et al., 2008). Therefore, urease is involved in N remobilization as well as in primary N assimilation. Plant ureases and arginases are housekeeping enzymes found in many if not all plant species (Witte and Medina-Escobar, 2001; Brownfield et al., 2008). Urease is a nickel metalloenzyme that in Arabidopsis (Arabidopsis thaliana) requires three urease accessory proteins (UAPs; AtUreD, AtUreF, and AtUreG) for activation (Witte et al., 2005a). Studies in bacteria demonstrated that UAPs form a complex with apo-urease and are required for posttranslational Lys carboxylation of apo-urease and the subsequent incorporation of two nickel ions into the active center. After activation, the UAPs dissociate from urease. The exact molecular function of each accessory protein in this process is not yet understood (Carter et al., 2009). Like urease, arginase is a metalloenzyme. It is best activated by manganese (Carvajal et al., 1996; Hwang et al., 2001), not requiring accessory proteins for activation.Urea plays an important role in agriculture because it is the most used N fertilizer worldwide (http://www.fertilizer.org/ifa), intensively employed in Asia for the cultivation of rice. Urea N partly reaches the plant as ammonium or nitrate because the fertilizer is already degraded in the environment by microbial ureases and may then be subject to nitrification. Alternatively, plants are capable of taking up urea from fertilization directly and assimilate its N (Kojima et al., 2007; Wang et al., 2008). Although rice is a major crop plant and rice production is heavily dependent on urea fertilization, the enzymes and the corresponding genes involved in rice urea metabolism have not yet been investigated. In this study, we identified the genes and cloned the corresponding cDNAs coding for rice arginase, urease, and the UAPs UreD, UreF, and UreG. The functionality of the corresponding proteins was demonstrated and biochemical parameters were determined. The general gene and protein structure of plant UreD and UreF were investigated and a direct interaction of rice UreD with apo-urease was discovered, leading to a refinement of the mechanistic view of plant urease activation. In physiological experiments, rice urease and arginase activities showed no significant response to different N-fertilizing regimes, while the amino acid composition in urea-grown plants was strongly imbalanced, indicating that urea N disturbs plant metabolism downstream of N assimilation.     

Characterization of PINK1 (PTEN-induced Putative Kinase 1) Mutations Associated with Parkinson Disease in Mammalian Cells and Drosophila

Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.     

Purification and Characterization of Arginine Decarboxylase from Soybean (Glycine max) Hypocotyls

Arginine decarboxylase (EC 4.1.1.19 [EC] ) was purified from soybean,Glycine max, hypocotyls by a procedure which includes ammoniumsulfate fractionation, acetone precipitation, gel filtrationchromatography, and affinity chromatography. Using this procedure,ADC was purified to one band in non-denaturing PAGE. The purifiedADC has an M_r of 240 kDa based on gel filtration chromatographyand is a trimer of identical subunits which has an estimatedM_r of 74 kDa based on SDS-PAGE. ADC is active between 30 and50°C and has a K_m value of 46.1 µM. ADC is very sensitiveto agmatine or putrescine but not to spermidine or spermine.In the presence of 0.5 mM agmatine (or putrescine), the enzymeactivity was inhibited by 70%. However, at the same concentrationof spermidine (or spermine), the enzyme activity was inhibitedby only 10–20%. (Received April 2, 1997; Accepted August 18, 1997)