Synthesis of antisense oligonucleotides carrying modified 2-5A molecules at their 5'-termini and their properties

The synthesis of 8-methyladenosine-substituted 2-5A tetramers with hydroxyalkyl groups at the 5'-phosphates and the corresponding 2-5A-antisense chimeras is described. These oligonucleotides were synthesized by the phosphoramidite method with a DNA/RNA synthesizer. These 2-5A tetramers with hydroxyethyl and hydroxybutyl groups at their 5'-phosphates were more resistant to hydrolysis by alkaline phosphatase than those without the hydroxyalkyl groups. Incorporation of the hydroxyethyl group into the 2-5A tetramer and 2-5A-antisense chimera slightly reduced the abilities of their analogues to activate recombinant human RNase L, but the abilities of the 2-5A tetramer and the 2-5A-antisense chimera both with the hydroxyethyl group and 8-methyladenosine returned to 80 and 50% relative to those of the oligonucleotides without the hydroxyethyl group and 8-methyladenosine, respectively. Furthermore, the enzyme activated by 8-methyladenosine-substituted 2-5A-antisense chimera with the hydroxyethyl group cleaved the complementary RNA as efficiently as that activated by 2-5A-antisense chimera without the hydroxyethyl group and 8-methyladenosine. Thus, the 2-5A-antisense chimera carrying the hydroxyethyl group and 8-methyladenosine will be a candidate for a novel antisense molecule.     

In vivo gene gun-mediated DNA delivery into rodent brain tissue

Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain.     

Mechanism of alpha-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of alpha-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (approximately 100 microM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.     

Sld2, Which Interacts with Dpb11 in Saccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

The DPB11 gene, which genetically interacts with DNA polymerase II (), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

How selection affects phenotypic fluctuation

The large degree of phenotypic fluctuation among isogenic cells highlighted by recent studies on stochastic gene expression confers fitness on some individuals through a ‘bet‐hedging’ strategy, when faced with different selective environments. Under a single selective environment, the fluctuation may be suppressed through evolution, as it prevents maintenance of individuals around the fittest state and/or function. However, as fluctuation can increase phenotypic diversity, similar to mutation, it may contribute to the survival of individuals even under a single selective environment. To discuss whether the fluctuation increases over the course of evolution, cycles of mutation and selection for higher GFP fluorescence were carried out in Escherichia coli. Mutant genotypes possessing broad GFP fluorescence distributions with low average values emerged under strong selection pressure. These ‘broad mutants’ appeared independently on the phylogenetic tree and increased fluctuations in GFP fluorescence were attributable to the variance in mRNA abundance. In addition to the average phenotypic change by genetic mutation, the observed increase in phenotypic fluctuation acts as an evolutionary strategy to produce an extreme phenotype under severe selective environments.     

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.     

RAD18 Activates the G2/M Checkpoint through DNA Damage Signaling to Maintain Genome Integrity after Ionizing Radiation Exposure

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.     

Clinical Features and Treatment Modes of Mandibular Fracture at the Department of Oral and Maxillofacial Surgery,Shimane University Hospital,Japan

Background

The number of elderly patients with maxillofacial trauma is rapidly increasing due to active lifestyles and longevity. Shimane prefecture has the fastest growing proportion of elderly individuals in Japan. The aim of this study was to reveal the distinctive features and treatment modes of mandibular fracture treatment mode in patients requiring hospitalization at the Department of Oral and Maxillofacial Surgery, Shimane University Hospital, Japan.

Patients and Methods

Patient age, sex, period between injury and first consultation, years since injury, cause of injury, fracture site, treatment, and duration of hospitalization were evaluated. Univariate Poisson regression, relative risk with 95% confidence interval based on the Wald test, Fisher’s exact test, and Kruskal-Wallis test were used to explore associations among clinical and demographic variables.

Results

In total, 305 patients were diagnosed with and hospitalized for mandibular fracture from 1980 to 2010. Younger age increased the risk for mandibular fracture. Incidence was higher in males than females, particularly in the young, but the male to female ratio decreased with age. The period until first hospital consultation decreased progressively over the study period. Fall was a much more frequent cause in patients aged ≥60 than in those aged <60 years. Mandibular fracture with condyle, symphysis, and angle involvement were most common and were associated with sex, age, and treatment mode. Length of hospitalization has decreased since 1980.

Conclusion

In our department, patients aged ≥60 years accounted for a greater proportion of mandibular fracture cases than in many previous studies, reflecting the greater proportion of elderly residents in Shimane prefecture.