Introduction of an Azido Group to the C-6 Position of Uridine by the Use of a 6-Iodouridine Derivative

Abstract

Nucleophilic addition-elimination reaction of azide ion to 6-iodo-2′,3′-O-isopropylidene-5′-O-methoxy-methyluridine proceeded under mild conditions to give a 6-azidouridine derivative.     

2-Deoxy-D-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. 2-Deoxy-d-ribose inhibited hypoxia-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not c-jun NH(2)-terminal kinase/stress-activated protein kinase in human leukemia HL-60 cells. 2-Deoxy-d-ribose also suppressed the levels of Bax attached to mitochondria under hypoxic conditions. SB203580, a specific inhibitor of the p38 MAPK, suppressed the hypoxia-induced apoptosis of HL-60 cells. These findings suggest that one of the molecular bases for resistance to hypoxia-induced apoptosis conferred by 2-deoxy-d-ribose is the inhibition of the p38 signaling pathway. The expression levels of TP are elevated in many malignant solid tumors and thus the 2-deoxy-d-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

Ovotransferrin is a redox-dependent autoprocessing protein incorporating four consensus self-cleaving motifs flanking the two kringles

Embryos of avian eggs and mammals are highly sensitive to oxidative stress and hence maintaining a steady reducing environment during the embryonic development is known to confer protection. Although information is completely lacking, proteins of avian egg albumin which have been suggested to play various biological functions, are the major targets for such reducing state during embryogenesis. In this study, we found that ovotransferrin (OTf), the second major protein in egg albumin, undergoes autocleavage at distinct sites upon reduction with thiol-reducing agent or thioredoxin-reducing system. Mass spectral and microsequencing analysis indicated that OTf is able to cleave itself through the unique chemical reactivity of four tripeptides motifs, HTT (residues 209-211), HST (residues 542-544) and two CHT (residues 115-117 and 454-456). Intriguingly, these self-cleavage sites were uniquely located upstream and downstream of the two disulfide kringle domains (residues 115-211 and 454-544) of OTf. These reduction-scissile sequences, His/Cys-X-Thr, are evolutionary conserved self-cleavage motifs found in several autoprocessing proteins including hedgehog proteins. Interestingly, reduction of other two members of transferrin family induced autocleavage patterns, similar to that of OTf, in bovine lactoferrin (bLf) while human lactoferrin (hLf) showed much less self-cleaving activity. This finding is the first to describe that transferrins are a new subset in the class of proteins able to carry out autoprocessing, providing insight into this unusual biochemical process that appears to be a molecular switch involved in triggering a yet unidentified function(s) of OTf as well as bLf.     

Expression of ADP-ribosylation factor-like protein 8B mRNA in the brain is down-regulated in mice fed a high-fat diet

A differential display was performed to analyze differential gene expression in the brains of mice in association with dietary high beef-tallow. Consumption of a high beef-tallow diet downregulated the expression of ADP-ribosylation factor-like protein 8B (Arl8B) mRNA in the brain. Arl8B mRNA was widely expressed in the mouse brain, including primary neuronal cells. The current study indicates that green fluorescent protein-fused Arl8B protein accumulated at the growth cones in primary neuronal cells, and that protrusions of human embryonic kidney 293 (HEK293) cells were significantly elongated by overexpression of Arl8B, suggesting an important role of Arl8B in neurite formation.     

A Simple PCR Method for Rapid Genotype Analysis of the TH-MYCN Transgenic Mouse

Background

The TH-MYCN transgenic mouse is the most widely used murine model of human neuroblastoma, in which a human MYCN oncogene is targeted to neuroectodermal cells of developing mice under the influence of the rat tyrosine hydroxylase promoter. So far, homozygous transgenic mice have been identified by either Southern blot or quantitative real-time PCR.

Principal Findings

To establish a simple and reliable genotyping method by conventional PCR, we confirmed the integration of the transgene in the TH-MYCN transgenic mouse by Southern blot and inverse PCR analyses. Our results showed that either five or six copies were found to be inserted in a head-to-tail tandem configuration at a single locus. The MYCN transgene/host DNA junction was sequenced and the integration site was identified at chromosome 18qE4. Finally, we succeeded in designing rapid, simple and reliable genotyping method by common PCR using primers flanking the integrated TH-MYCN transgene.

Conclusion

We established a simple and reliable genotyping PCR method for determining the integration site of the TH-MYCN transgene that enables all possible genotypes to be distinguished within several hours. TH-MYCN mice are excellent model for human neuroblastoma study, thus our results will largely be useful for facilitating the pace of neuroblastoma study, including in the study of the tumourigenic process, and in the development of therapies to treat patients suffering from neuroblastoma.     

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.     

Multiple-color fluorescence imaging of chromosomes and microtubules in living cells

Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with alpha-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.