Agrobacterium-mediated transformation of élite indica and japonica rice cultivars

A rapid, efficient, routine system has been established forAgrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1. Calli induced from embryos of mature rice seeds were cocultivated withA. tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system. Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively. T1 seeds were harvested within 7–8 mo of initiation of mature embryo cultures. Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers. Mendelian inheritance of the transgenes was confirmed in T1 progeny.     

A novel dual peroxisome proliferator-activated receptors α and γ agonist with beneficial effects on insulin resistance and lipid metabolism

Peroxisome proliferator-activated receptors (PPARs) α and γ are key regulators of lipid homeostasis and insulin resistance. In this study␣we show that a novel compound, 3-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]-phenyl}- 2-[2-(2-nitro-phenoxy)-acetyl amino]-propionic acid (O325H), is an agonist with dual effect on PPARα/γ by using dual-luciferase reporter gene assay. By activating PPARα and PPARγ simultaneously, O325H promotes pre-adipocyte differentiation and up-regulates the expression of glucose and lipid metabolic target genes. In diabetic mice, administration of O325H at 10 mg/kg decreases the blood lipid and glucose levels. Therefore, O325H has dual action on PPARα and PPARγ and is a promising agent for the amelioration of lipid metabolic disorders and diabetes associated with insulin resistance.     

Supramolecular architectures based on di-, tri- and tetrameric complexes with N,N′-diacylhydrazide ligands

Five new complexes, [Co₃(HL₁)₂(Py)₈]·4CH₃OH (1), [Ni₃(HL₁)₂(Py)₄]₂·2DMF (2), [Co₃(H₂L₂)₂(Py)₈]·2NO₃ (3), [Ni₂(HL₂)(Py)₆] (4) and [Cu₄(HL₂)₂(Py)₄]·4DMF (5) (H₄L₁ = N-propionyl-4-hydroxysalicylhydrazide, H₄4-hopshz; H₅L₂ = N-(3-carboxy-cis-2-propenoyl)-4-hydroxysalicylhydrazide, H₅4-hocpshz) have been obtained from two N,N′-diacylhydrazide ligands and characterized by elemental analysis, FT-IR, X-ray diffraction and antimicrobial activities. These di-, tri-, and tetrameric complexes are connected into three-dimensional supramolecular architectures with interesting topologies through O-H?O, C-H?O and C-H?π interactions. 1-3 are linear trimeric complexes with the ligands triply-deprotonated. Topological analysis indicates that they exhibit 2D (4,4), 3D (6,8)-connected (3³4⁹5²6)(3⁴4¹²5⁷6⁴7) and 8-connected (4²⁵6³) net, respectively. 4 and 5 possess dimeric and tetrameric structures, which are extended into 7-connected (3³4¹³5³6) and 4-connected (4,4) net, respectively.     

Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine–arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.DNA double-stranded breaks (DSBs)³ can be produced in physiological and genotoxic processes. Improper repair or failure to repair DSBs can lead to gene deletions, duplications, translocations, and missegregation of large chromosome fragments, which may result in gene dosage imbalance, cancer development, or cell death (1–3). Historically, two distinct pathways have been described which ensure that DSBs are repaired: nonhomologous end joining (NHEJ) and homologous recombination (HR). During HR, the damaged chromosome interacts via synapsis with an undamaged DNA molecule with which it shares extensive sequence homology, usually its sister chromatid (4, 5). HR is most active in the late S and G₂ phases of the cell cycle. In contrast, NHEJ is active throughout the cell cycle and requires little or no DNA homology during repair; thus, it is traditionally considered an error-prone repair pathway (6, 7). However, accumulating evidence from recent studies suggests that there exists an error-free NHEJ subpathway (8, 9).Two types of end-joining reactions can be defined operationally. The first one, which may be called conservative NHEJ (C-NHEJ), is characterized by the precise joining of short, overhanging, complementary ends. Proteins including Ku70/Ku80 and XRCC4 (10–12) are associated with this highly efficient pathway, whereby most ends are rejoined successfully without any alteration of the DNA sequence (8). The alternative pathway for NHEJ is the highly mutagenic and deletional NHEJ (D-NHEJ), which results in short deletions after use of imperfect microhomology of about 1–10 bp at the repair junctions. D-NHEJ activity has been demonstrated in the budding yeast Saccharomyces cerevisiae. In addition, D-NHEJ is independent of Rad52, Rad1, or Ku80 but depends on Mre11 in yeast (13, 14). However, the genetic determinants of this subpathway have not been well established in mammalian cells.Mre11 is the core subunit of the Mre11·Rad50·Nbs1 complex (called the MRN complex), which is conserved throughout all kingdoms of life. The MRN complex is a central player in most aspects of the cellular response to DSBs, including HR, NHEJ, telomere maintenance, and DNA damage checkpoints (15–17). Loss of Mre11 results in increased radiosensitivity and chromosomal instability (17). Patients with germ line mutations of Mre11 have clinical presentations similar to those of ataxia telangiectasia patients (ataxia telangiectasia-like disorder) (18).After DNA damage, the MRN complex is recruited to the sites of damage via zinc hooks at the ends of the long, flexible arms of Rad50 (19, 20). Mre11 contains both single-stranded DNA endonuclease and 3′-5′ exonuclease activities in vitro, but in vivo Mre11 is also implicated in 5′-3′ DSB resection. The MRN complex also interacts with BRCA1 and CtIP, which may be essential for DSB end resection to generate 3′ overhanging single-stranded DNA during initiation of HR (21, 22).Mre11 has an N-terminal nuclease domain, which contains five phosphoesterase motifs, and a C-terminal glycine–arginine-rich domain (GAR). Arthur et al. (23) showed that an H85L mutation completely abrogated exonuclease activity, whereas binding to Rad50 and Nbs1 was retained. Complementation of ataxia telangiectasia-like disorder cells with this mutant, called Mre11-3, restored the localization of the MRN complex to DSBs in IR-induced foci (23, 24). Methylation of the GAR region has also been shown to be important for the DNA binding and exonuclease activity of Mre11 in vitro (25, 26). Both the crystal structure of yeast Mre11 and data from conditional knock-out mice (Mre11^H129N/Δ) reveal that the nuclease activity of Mre11 is required for HR repair of DSBs (22, 27). However, the role of Mre11 in NHEJ is not well defined (27, 28). Most recently, Mre11 was reported to support NHEJ in mammalian cell (29–31). However, whether Mre11 regulates both NHEJ subpathways or only D-NHEJ is controversial, and the mechanisms by which Mre11 is involved in NHEJ remain to be established.To address these questions, we have established a system that can analyze the accuracy and efficiency of rejoining of two adjacent DSB ends at chromosomal level in human embryonic kidney 293 (HEK293) cells. We show here that Mre11 siRNA knockdown in these cells results in significant reduction of the overall NHEJ efficiency. Upon sequencing the repair junctions, we found that Mre11 siRNA knockdown suppressed D-NHEJ by ∼10-fold, reflected by a reduction of small deletions in the repair junction, but it had no effect on the efficiency of C-NHEJ. Mutation of Mre11 in either the phosphoesterase domain (Mre11-3) or the GAR region (Mre11-R/A) to produce abnormal exonuclease activity impaired the D-NHEJ pathway only. The D-NHEJ deficiency is significantly more severe in cells with Mre11-R/A than that in cells with Mre11-3. Therefore, our data suggest that Mre11 is required specifically for D-NHEJ repair of DNA DSBs and that its exonuclease activity is at least one of the important mechanisms for this DNA end joining subpathway.     

A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.     

Validation and application of an LC–ESI-MS method for simultaneous determination of astilbin and its major metabolite 3′-O-methylastilbin in rat plasma

We herein describe the development of an LC–MS method for simultaneous determination of astilbin and 3′-O-methylastilbin in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts on to a Shim-pack C₁₈ column (150 mm × 2.0 mm I.D., 5 μm) with gradient elution and detection in negative ionization mode. Initially, the method was validated regarding linearity, accuracy and precision. The correlation coefficients of all the calibration curves showed good linearity (r > 0.999) within test ranges, and the relative deviation was less than 10% for intra- and inter-day assays. Besides, this method was also validated for its stability, extraction efficiency, matrix effect and so on. Finally, this proposed method was successfully applied to rat pharmacokinetic study and yielded the most comprehensive data on systemic exposure of them to date.