Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4

The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.     

Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival

Engagement of the FcepsilonRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3' position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34(+) peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcepsilonRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcepsilonRI-responsiveness.     

Screening of siRNA target sequences by using fragmentized DNA

BACKGROUND: RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms. In mammals, RNAi can be induced by using short interfering RNA (siRNA). The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences. METHODS: We developed an siRNA target sequence selection system by first constructing parallel-type siRNA expression vector libraries carrying siRNA expression fragments originating from fragmentized target genes, and then using a group selection system. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes. RESULTS: We carried out the first screening of groups containing more than 100 random siRNA expression plasmids in total for each target gene, and successfully obtained target sequences with very strong efficacy. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity. CONCLUSIONS: This system should allow us to perform screening for powerful target sequences, by including all possible target sequences for any gene, even without knowing the whole sequence of the target gene in advance. At the same time, target sequences that should be avoided due to cytotoxicity can be identified.     

Hypothalamic cardiovascular effects of angiotensin-(1-7) in spontaneously hypertensive rats

The objective of the present work was to study the cardiovascular actions of the intrahypothalamic injection of Ang-(1-7) and its effects on the pressor response to Ang II in spontaneously hypertensive (SH) rats and Wistar Kyoto (WKY) animals. In anaesthetized SH and WKY rats, a carotid artery was cannulated for mean arterial pressure (MAP) measurement and a stainless-steel needle was inserted into the anterior hypothalamus for drug administration. The cardiovascular effects of the intrahypothalamic administration of Ang-(1-7) were determined in SH and WKY rats. In SH rats, the effect of irbesartan and D-Ala-Ang-(1-7) on Ang-(1-7) cardiovascular effect was also evaluated. Ang II was administered in the hypothalamus of SH and WKY rats and changes in blood pressure and heart rate were measured followed by the administration of Ang II, Ang II+Ang-(1-7) or Ang II+D-Ala-Ang-(1-7). Ang-(1-7) did not the change basal MAP in WKY rats, but induced a pressor response in SH animals. Whilst the co-administration of D-Ala-Ang-(1-7) did not affect the response to Ang-(1-7), the previous administration of irbesartan prevented the effect of the peptide. The intrahypothalamic injection of Ang II induced a significantly greater pressor response in SH animals compared to normotensive rats. The co-administration of Ang-(1-7) with Ang II did not affect the pressor response to Ang II in the WKY group. In SH rats, whilst the co-administration of Ang-(1-7) with Ang II reduced the pressor response to Ang II, the concomitant application of D-Ala-Ang-(1-7) with Ang II increased the pressor response to the octapeptide after 5 and 10 min of intrahypothalamic administration. In conclusion, our result demonstrated that the biologically active peptide Ang-(1-7) did not participate in the hypothalamic blood pressure regulation of WKY animals. In SH rats, Ang-(1-7) exerted pleiotropic effects on blood pressure regulation. High dose of the heptapeptide produced a pressor response because of an unspecific action by activation of AT1 receptors. The concomitant administration of lower doses of Ang-(1-7) with Ang II reduced the pressor response to the octapeptide. Finally, the effect of AT(1-7) antagonist on Ang II pressor response suggested that hypothalamic formed Ang-(1-7) are implicated in the regulation of the cardiovascular effects of Ang II.     

Working at the cutting edge: the creation of allosteric ribozymes

The appropriate folding of catalytic RNA is a prerequisite for effective catalysis. A novel ribozyme, the maxizyme, has been generated and its activity can be controlled allosterically. The maxizymes work both in vitro and in vivo indicating the potential utility of this novel class of ribozyme as a gene-inactivating agent with a biosensor function.     

Caldesmon regulates axon extension through interaction with myosin II

To begin the process of forming neural circuits, new neurons first establish their polarity and extend their axon. Axon extension is guided and regulated by highly coordinated cytoskeletal dynamics. Here we demonstrate that in hippocampal neurons, the actin-binding protein caldesmon accumulates in distal axons, and its N-terminal interaction with myosin II enhances axon extension. In cortical neural progenitor cells, caldesmon knockdown suppresses axon extension and neuronal polarity. These results indicate that caldesmon is an important regulator of axon development.     

Positive selection of Toll-like receptor 2 polymorphisms in two closely related old world monkey species, rhesus and Japanese macaques

Toll-like receptor 2 (TLR2) plays an important role in the recognition of a variety of pathogenic microbes. In the present study, we compared polymorphisms of TLR2 locus in two closely related old world monkey species, rhesus monkey (Macaca mulatta) and Japanese monkey (Macaca fuscata). By nucleotide sequencing of the third exon of TLR2 gene from 21 to 35 respective individuals, we could assign 17 haplotype combinations of 17 coding SNPs of ten non-synonymous and seven synonymous substitutions. A non-synonymous substitution at codon position 326 appeared to be differentially fixed in each species, asparagine for M. mulatta whereas tyrosine for M. fuscata, and may contribute to certain functional properties because it locates in the region contributing to ligand binding and interaction with dimerization partner of TLR2-TLR1 heterodimeric complex. Although TLR2 alleles have diverged to similar extent in both species, they have evolved in significantly different ways; TLR2 of M. fuscata has undergone purifying selection while the membrane-proximal part of the extracellular domain of M. mulatta TLR2 exhibits higher rates of non-synonymous substitutions, indicating a trace of Darwinian positive selection.