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.     

Inhibition of NF-{kappa}B improves left ventricular remodeling and cardiac dysfunction after myocardial infarction

Several studies have demonstrated that NF-kappaB is substantially involved in the progression of cardiac remodeling; however, it remains uncertain whether the continuous inhibition of NF-kappaB is effective for the prevention of myocardial remodeling. Myocardial infarction (MI) was produced by ligation of the left anterior coronary artery of rats. IMD-0354 (10 mg/kg per day), a novel phosphorylation inhibitor of IkappaB that acts via inhibition of IKK-beta, was injected intraperitoneally starting 24 h after induction of MI for 28 days. After 28 days, the IMD-0354-treated group showed significantly improved survival rate compared with that of the vehicle-treated group (P < 0.05). Although infarct size was similar in both groups, improved left ventricular (LV) remodeling and diastolic dysfunction, as indicated by smaller LV cavity (LV end-diastolic area: vehicle, 74.13 +/- 3.57 mm(2); IMD-0354, 55.00 +/- 3.73 mm(2); P < 0.05), smaller peak velocity of early-to-late filling wave (E/A) ratio (vehicle, 3.87 +/- 0.26; IMD-0354, 2.61 +/- 0.24; P < 0.05), and lower plasma brain natriuretic peptide level (vehicle, 167.63 +/- 14.87 pg/ml; IMD-0354, 110.75 +/- 6.41 pg/ml; P < 0.05), were observed in the IMD-0354-treated group. Moreover, fibrosis, accumulation of macrophages, and expression of several factors (transforming growth factor-beta1, monocyte chemoattractant protein-1, matrix metalloproteinase-9 and -2) in the noninfarcted myocardium was remarkably inhibited by IMD-0354. In conclusion, inhibition of NF-kappaB activation may reduce the proinflammatory reactions and modulate the extracellular matrix and provide an effective approach to prevent adverse cardiac remodeling after MI.     

Fractionated irradiation improves the mating performance of the West Indian sweet potato weevil Euscepes postfasciatus

• 1 The sterile insect technique (SIT) is widely used to suppress or eradicate target pest insect populations.
• 2 The effectiveness of SIT depends on the ability of released sterile males to mate with and inseminate wild females. The use of gamma radiation to induce sterility, however, negatively affects both somatic cells as well as reproductive cells. Consequently, mating performance of sterilized individuals decreases drastically over time. The mating propensity of sterilized Euscepes postfasciatus (Fairmaire) males irradiated with a single dose of 150 Gy (the current standard of the Okinawa Prefecture SIT programme) is equal to that of non‐irradiated weevils for the first 6 days.
• 3 Fractionated irradiation, in which a sterilizing dose is delivered over time in a series of smaller irradiations, reduces the damage of irradiation in insects. In the present study, we evaluated the effect of fractionated irradiation on male fertilization ability, longevity and mating propensity of E. postfasciatus for a period of 16 days after irradiation.
• 4 Although fractionated irradiation totalling 150 Gy was found to induce full sterility regardless of the number of individual doses, the mating propensity of male weevils sterilized by fractionated irradiation was maintained for the first 12 days. These results demonstrate that fractionated irradiation can be highly advantageous in programmes aimed at eradication of E. postfasciatus.

     

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.     

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.     

PPAR(alpha) and PPAR(gamma) activators suppress the monocyte-macrophage apoB-48 receptor

Certain triglyceride-rich lipoproteins (TRLs), specifically chylomicrons, dyslipemic VLDLs, and their remnants, are atherogenic and can induce monocyte-macrophage foam cell formation in vitro via the apolipoprotein B-48 receptor (apoB-48R). Human atherosclerotic lesion foam cells express the apoB-48R, as determined immunohistochemically, suggesting it can play a role in the conversion of macrophages into foam cells in vivo. The regulation of the apoB-48R in monocyte-macrophages is not fully understood, albeit previous studies indicated that cellular sterol levels and state of differentiation do not affect apoB-48R expression. Since peroxisome proliferator-activated receptors (PPARs) regulate some aspects of cellular lipid metabolism and may be protective in atherogenesis by up-regulation of liver X-activated receptor alpha and ATP-binding cassette transporter A1, we examined the regulation of apoB-48R by PPAR ligands in human monocyte-macrophages. Using real-time PCR, Northern, Western, and functional cellular lipid accumulation assays, we show that PPARalpha and PPARgamma activators significantly suppress the expression of apoB-48R mRNA in human THP-1 and blood-borne monocyte-macrophages. Moreover, PPAR activators inhibit the expression of the apoB-48R protein and, notably, the apoB-48R-mediated lipid accumulation of TRL by THP-1 monocytes in vitro. If PPAR activators also suppress the apoB-48R pathway in vivo, diminished apoB-48R-mediated monocyte-macrophage lipid accumulation may be yet another antiatherogenic effect of the action of PPAR ligands.     

The hedgehog signal induced modulation of bone morphogenetic protein signaling: an essential signaling relay for urinary tract morphogenesis

Background

Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases.

Methodology/Principal Findings

To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh^−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells.

Conclusions/Significance

This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh-responsive mesenchymal Bmp signaling maintains the population of peri-cloacal mesenchyme cells, which is essential for the development of the ureter and the upper urinary tract.     

Synthesis of glyceroyl beta-N-acetyllactosaminide and its derivatives through a condensation reaction by cellulase

A condensation reaction between N-acetyllactosamine and glycerol was directly catalyzed by using a commercially available cellulase preparation from Trichoderma reesei. 1-O-beta-N-Acetyllactosaminyl-(R, S)-glycerols (1) were readily synthesized in a 5% yield based on the N-acetyllactosamine added and conveniently isolated by two-step column chromatographies. The use of a partially purified enzyme increased 2.3-fold the yield of 1, compared to that of the crude enzyme containing beta-D-galactosidase activity. When various alkanols (n:2-4) were used in the condensation reaction, the corresponding alkyl beta-N-acetyllactosaminides were obtained in yields of 0.3-1.1% of the desired compounds.     

Microphthalmia (mi) mice display an aberrant bone trace element composition

It has been recognized that bone trace element composition analysis provides clues when analyzing bone-related physiological conditions. Increasing numbers of bone-related genetic diseases have been identified recently. In this study, we have analyzed bone trace element composition in a genetic mutant animal model. Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types, including melanocytes, mast cells, and osteoclasts. Previous studies have shown that different alleles of the mi locus show osteopetrosis. In order to gain insights into the effects of a particular genetic defect on bone trace element composition and bone structure, we performed bone trace element composition analysis using inductively coupled plasma atomic emissions spectrometry (ICP-AES). Marked changes in bone trace element levels were found in vertebrate bones of mi mutant mice. The implications and possible applications of bone trace element analysis will be discussed in this article.