A stepwise model for double-stranded RNA processing by ribonuclease III

RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family represented by bacterial RNase III and eukaryotic Rnt1p, Drosha and Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the family. Previously, we have shown that RNase III uses two catalytic sites to create the 2-nucleotide (nt) 3' overhangs in its products. Here, we present three crystal structures of RNase III in complex with double-stranded RNA, demonstrating how Mg(2+) is essential for the formation of a catalytically competent protein-RNA complex, how the use of two Mg(2+) ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modelled a protein-substrate complex and a protein-reaction intermediate (transition state) complex on the basis of the crystal structures. Together, the crystal structures and the models suggest a stepwise mechanism for RNase III to execute the phosphoryl transfer reaction.     

Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs

Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.     

Transgene integration and organization in Cotton (Gossypium hirsutum L.) genome

While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement.     

Hand preference during unimanual and bimanual reaching actions in Sichuan snub-nosed monkeys (Rhinopithecus roxellana)

Hand preferences were investigated during one unimanual action (food-reaching) and one bimanual action (mount-reaching) in a semi-free-ranging group of Sichuan snub-nosed monkeys (Rhinopithecus roxellana) in Zhouzhi National Nature Reserve, Qinling Mountains of China. Nine of 14 individuals tested on the unimanual food-reaching action and all six individuals tested on the bimanual mount-reaching action exhibited a manual preference. Both significant right- and left-handed preferences were observed in the two actions. Sex did not affect either direction or strength of hand preference in the unimanual action. Hand preference for the bimanual action was stable over time, and the strength of hand preference was significantly stronger in the bimanual action than in the unimanual action.     

Reproductive parameters of wild female Rhinopithecus roxellana

On the basis on 6 years of observation, we estimated the reproductive parameters of a Golden snub-nosed monkey (Rhinopithecus roxellana) group in the Qinling Mountains, China. We observed 88 births in 47 females from 2001 to 2006. Two methods were used to calculate the birthrate. The first method is based on the number of births observed in a year, giving 0.49+/-0.07 (mean+/-SD), and the second method is based on the female-years of observation, giving 0.49+/-0.17 births per female per year in this troop. The mean interbirth interval is 21.88+/-6.01 months (mean+/-SD). The mortality of infant born between 2002 and 2005 was 22.4%. The interbirth intervals of females that had lost an infant before the age of 6 months were significantly shorter than that of females whose infants survived for more than 6 months. A female usually gives birth once every 2 years if the previous offspring survives to a weaning age of 5-6 months, or will give birth in the next year if the previous young dies before reaching an age of 6 months. Births were significantly concentrated during March to May of each year. The mean birth date was on April 14, median was April 12; and the standard deviation was 13.98 days. Birth peak occurs 6-7 months after mating peak. From observations on 15 individuals that gave birth for the first time, we concluded that the wild female Golden snub-nosed monkeys in Qinling Mountains start giving birth at an age of 5 or 6 years. We suggest that the seasonal reproductive pattern is an adaptive response to the availability of seasonal food. Our results are consistent with the hypothesis that these reproductive characteristics are a result of adaptation to the seasonality of mountain climate and food resources.     

Tricationic pyridium porphyrins appending different peripheral substituents: experimental and DFT studies on their interactions with DNA

Four tricationic pyridium porphyrins appending hydroxyphenyl, methoxyphenyl, propionoxyphenyl or carboxyphenyl group at meso-20-position of porphyrin core have been synthesized and their abilities to bind and cleave DNA have been investigated. Using a combination of absorption, fluorescence, circular dichroism (CD) spectra, thermal DNA denaturation as well as viscosity measurements, their binding modes and intrinsic binding constants (K_b) to calf DNA (CT DNA) were comparatively studied and also compared with those of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP). The results suggest that the K_b values of these porphyrins are greatly influenced by the number of positive charges and steric hindrance. Theoretical calculations applying the density functional theory (DFT) have been carried out and explain their DNA-binding properties reasonably. The efficiency of DNA photocleavage by these porphyrins shows high dependence on the values of K_b.     

Gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides by cDNA microarrays

To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy.     

Nicotinamide attenuates retinal ischemia and light insults to neurones

The aim of the present studies was to determine whether nicotinamide is effective in blunting the negative influence of ischemia/reperfusion to the rat retina in situ and of light to transformed retinal ganglion cells (RGC-5 cells) in culture. Ischemia was delivered to the retina of one eye of rats by raising the intraocular pressure. Nicotinamide was administered intraperitoneally just before ischemia and into the vitreous immediately after the insult. Electroretinograms (ERGs) of both eyes were recorded before and 5 days after ischemia. Seven days after ischemia, retinas were analysed for the localization of various antigens. Retinal and optic nerve extracts were also prepared for analysis of specific proteins and mRNAs. Also, RGC-5 cells in culture were given a light insult (1000 lux, 48 and 96 h) and evidence for reduced viability and apoptosis determined by a variety of procedures. Nicotinamide was added to some cultures to see whether it reversed the negative effect of light. Ischemia/reperfusion to the retina affected the localization of Thy-1, neuronal nitric oxide synthase (NOS) and choline acetyltransferase (ChAT), the a- and b-wave amplitudes of the ERG, the content of various retinal and optic nerve proteins and mRNAs. Significantly, nicotinamide statistically blunted many of the effects induced by ischemia/reperfusion which included the activation of poly-ADP-ribose polymerase (PARP). Light-induced apoptosis of RGC-5 cells in culture was attenuated by nicotinamide and the PARP inhibitor NU1025. The presented data show that nicotinamide attenuates injury to the retina and RGC-5 cells in culture caused by ischemia/reperfusion and by light, respectively. Evidence is provided to suggest that nicotinamide acts as a PARP inhibitor and possibly an antioxidant.     

Hormone interactions to Leu-rich repeats in the gonadotropin receptors. II. Analysis of Leu-rich repeat 4 of human luteinizing hormone/chorionic gonadotropin receptor

The luteinizing hormone receptor (LHR) consists of an approximately 350-amino acid-long N-terminal extracellular exodomain and a membrane-associated endodomain of similar size. Human chorionic gonadotropin (hCG) binds to the exodomain, and then hCG/exodomain complex is thought to make a secondary contact with the endodomain and generate hormone signals. The sequence alignment of the exodomain shows imperfectly matching eight to nine Leu-rich repeats (LRRs). In the preceding article (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435), we have shown that LRR2 and LRR4 are crucial for hormone binding. In this work, we have examined the residues of LRR4, in particular Leu(103) and Ile(105) in the putative beta strand. Our data show that Leu(103) and Ile(105) are involved in the specific, hydrophobic interaction of the LRR4 loop, likely to form the hydrophobic core. This loop is crucial for the structural integrity of all of the LRRs. In contrast, the downstream sequence consisting of Asn(107), Thr(108), Gly(109), and Ile(110) of LRR4 is crucial for cAMP induction but not for hormone binding, folding, and surface expression. This implicates, for the first time, its involvement in the interaction with the endodomain and signal generation. The evidence for the interaction is presented in the following article.