U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells

The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.     

Endothelial NO Synthase (eNOS) phosphorylation regulates coronary diameter during ischemia-reperfusion in association with oxidative stress

The link between endothelial nitric oxide synthase (eNOS) activation and vascular diameter during ischemia-reperfusion was investigated in the rat heart. After short (<30 min) and long (>45 min) time of ischemia conferred by coronary artery occlusion of the rats, reperfusion caused dilatation and constriction of arterioles, respectively. Partial oxygen pressure (pO2) measurement of the heart by the electrode confirmed the hyper-perfusion and no-reflow phenomena during reperfusion, as well as myocardial ischemia. The vascular diameter was correlated with phosphorylation of Akt and serine 1177 residue of eNOS, and formation of NO-bound guanylate cyclase (GC) by immuoflorescence study. Western blotting confirmed the phosphorylation of eNOS-Ser1177 depending on ischemia time. The constriction during reperfusion after 45 min of ischemia is supposedly caused by the inhibition of Akt-mediated eNOS-Ser1177 phosphorylation, which was suppressed by a PKC inhibitor chelerythrine, or ROS scavengers N-2-mercaptopropionyl glycine (MPG) and 4,5-Dihydroxy-1, 3-benzenedisulfonic acid disodium salt (Tiron). However, an endothelin receptor antagonist BQ123 alleviated the vasoconstriction by increasing NO availability but not eNOS-Ser1177 phosphorylation. Thus, vascular patency is correlated with eNOS-Ser1177 phosphorylation in association with ROS, and PKC during reperfusion. Endothelin inhibits vasodilatation by reducing NO availability during reperfusion.     

Detection of Short Protein Coding Regions within the Cyanobacterium Genome: Application of the Hidden Markov Model

The gene-finding programs developed so far have not paid muchattention to the detection of short protein coding regions (CDSs).However, the detection of short CDSs is important for the studyof photosynthesis. We utilized GeneHacker, a gene-finding programbased on the hidden Markov model (HMM), to detect short CDSs(from 90 to 300 bases) in a 1.0 mega contiguous sequence ofcyanobacterium Synechocystis sp. strain PCC6803 which carriesa complete set of genes for oxygenic photosynthesis. GeneHackerdiffers from other gene-finding programs based on the HMM inthat it utilizes di-codon statistics as well. GeneHacker successfullydetected seven out of the eight short CDSs annotated in thissequence and was clearly superior to GeneMark in this rangeof length. GeneHacker detected 94 potentially new CDSs, 9 ofwhich have counterparts in the genetic databases. Four of thenine CDSs were less than 150 bases and were photosynthesis-relatedgenes. The results show the effectiveness of GeneHacker in detectingvery short CDSs corresponding to genes.     

The promoter of rbcS in a C3 plant (rice) directs organ-specific,light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding -glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.     

Effects of a bout of traditional and original sumo training on neutrophil immune function in amateur university sumo wrestlers

In order to examine in detail the influence on the neutrophil immune function in sumo wrestlers of performing traditional and original training we examined changes in the neutrophil immune function in 17 male amateur university sumo wrestlers (aged 20.2 ± 1.5 years), before (‘Pre’) and after the training (‘Post’) for 2.5 h under fasting conditions. Assays included blood leukocyte and neutrophil counts, serum concentration of immunoglobulins, complements, myogenic enzymes and neutrophil oxidative burst activity (OBA) and phagocytic activity (PA). Myogenic enzymes, neutrophil counts, the ratio of neutrophil counts:leukocyte counts significantly increased and immunoglobulins and complements decreased in Post compared with Pre. There was a positive correlation between the change of neutrophil counts before and after the training and the change of creatine kinase (r = 0.667, p < 0.01). The Post OBA significantly increased and PA significantly decreased compared with Pre. It was concluded that sumo training causes muscular damage and an increase in the neutrophil count as a response. In this time, although OBA increased, PA decreased after training. Compensation between PA and reactive oxygen species production may exist to maintain the overall integrity of the neutrophil immune function. Copyright © 2008 John Wiley & Sons, Ltd.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

Settlement of Planulae of the Moon Jellyfish Aurelia aurita onto Hydrophilic Polycarbonate Plates Modified by Atmospheric Plasma Treatment

It has been reported that planula larvae of some jellyfish prefer artificial substrates for settlement. This research focused on the relationship between the settlement of planulae and the wettability of artificial substrate surfaces. We used atmospheric plasmas to change the wettability of the surfaces of polycarbonate (PC) plates because plasma treatment has no chemical side effects. The treatment made the surfaces hydrophilic, as evidenced by the decrease of contact angle from 85° to 35°. X-ray photoelectron spectroscopy revealed that the change of wettability of the PC plates could be attributed to N₂, which was probably ionized in the air above the plates. Scanning electron microscopy revealed no difference in the surface morphology of the plates before and after plasma treatment. Results of bioassays using treated PC plates showed that planulae tended to preferentially settle on hydrophobic surfaces.