Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.     

An Evidence for the Repair of Double-Strand Scissions in DNA of Micrococcus radiodurans after Alpha Bombardment

The conversion of prochaetoglobosins as plausible precursors into mycotoxin chaetoglobosin A (1) in a cell-free system of Chaetomium subaffine was unsuccessful. However, reductase activity of the 20-keto-analogues (1), and prochaetoglobosins II (5) and III (6) were found in a microsomal fraction of this fungi. Two new metabolites of chaetoglobosins, named chaetoglobosin F_ex (2) and 20-dihydro-chaetoglobosin A (3), were also isolated from the same micro-organisms. Their structures were elucidated by spectroscopic data and chemical transformation.     

Metabolism of Thiophene-2-carboxylate by a Photosynthetic Bacterium

A photosynthetic bacterium, which can grow photosynthetically on benzoate, was isolated from sewage mud. Various kinds of aromatic compounds including heterocyclic aromatic compounds were photometabolized by the washed cells grown photosynthetically on benzoate with no lag period. Among these, thiophene-2-carboxylate was metabolized most rapidly to its (+)-tetrahydro derivative. The same strain could also grow on succinate under photosynthetic conditions. However, thiophene-2-carboxylate was only photometabolized after a long lag period by the washed cells grown photosynthetically on succinate, and the metabolite was not its (+)-tetrahydro derivative but (+)-3-hydroxytetrahydrothiophene-2-carboxylate. In the presence of chloramphenicol, an inhibitor of protein synthesis, the photometabolism of thiophene-2-carboxylate by the washed cells grown photosynthetically on benzoate was not affected at all, but the photometabolism of the same substrate by the washed cells grown photosynthetically on succinate was completely inhibited. These results indicate that a reduction system of broad substrate specificity for aromatic rings is already present in the benzoate-grown cells but absent in the succinate-grown cells. It seems that such a reduction system for aromatic rings is induced by an aromatic substrate.     

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background

Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.

Methods and Results

IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.

Conclusions

Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.     

Phylogeography of lateral plate dimorphism in the freshwater type of ninespine sticklebacks, genus Pungitius

The freshwater type of ninespine sticklebacks, genus Pungitius, is widely distributed in northern Japan and reproductively isolated from other genetically divergent types endemic to small regions in Japan. This type expresses dimorphism in its lateral plate morphology: complete and partial row morphs. The two morphs show a parapatric distribution in Japan. To clarify the process involving the distribution of these two morphs, we examined their phylogeography based on restriction fragment length polymorphism of an entire mitochondrial DNA (mtDNA). The survey was carried out with seven restriction enzymes on the populations of the freshwater type collected from 41 localities across the distribution range in Japan, and 6 further Pungitius populations from the Okhotsk Sea coast of Russia were appended. An unweighted pair-group method with arithmetic averages (UPGMA) tree among 54 mtDNA haplotypes resolved eight clustering groups that differed in sequence divergence by approximately 1.3%–2.1%. Two of the eight groups were found only in Russia. mtDNA phylogenies constructed by neighbor-joining and Wagner parsimony methods suggested that the haplotypes of each plate morph were polyphyletic. The geographic distribution pattern of these groups suggests that they should be classified into two broad categories, one with extensive distribution and the other with localized distribution of the constituent haplotypes within a group. The former groups were found mainly in the populations with the completely plated morph and the latter groups with the partially plated morph. It is supposed that twice dispersals of dimorphic or complete plated ancestors and genetic differentiation during the interglacial played an important role in the formation of the present distribution of the two morphs in Japan. Received: March 28, 2000 / Revised: November 3, 2000 / Accepted: January 16, 2001     

A Randomized,Single-Blind,Placebo-Controlled Study on the Efficacy of the Arthrokinematic Approach-Hakata Method in Patients with Chronic Nonspecific Low Back Pain

Study design

cized, single-blind, controlled trial.

Objective

To investigate the efficacy of the Arthrokinematic approach (AKA)-Hakata (H) method for chronic low back pain.

Summary of Background Data

The AKA-H method is used to manually treat abnormalities of intra-articular movement.

Methods

One hundred eighty-six patients with chronic nonspecific low back pain randomly received either the AKA-H method (AKA-H group) or the sham technique (S group) monthly for 6 months. Data were collected at baseline and once a month. Outcome measures were pain intensity (visual analogue scale [VAS]) and quality of life (the Roland-Morris Disability Questionnaire [RDQ] and Short Form SF-36 questionnaire [SF-36]).

Results

At baseline, the VAS, RDQ, and SF-36 scores showed similar levels between the groups. After 6 months, the AKA-H group had more improvement in the VAS (42.8% improvement) and RDQ score (31.1% improvement) than the sham group (VAS: 10.4% improvement; RDQ: 9.8% improvement; both, P < 0.001). The respective scores for the SF-36 subscales (physical functioning, role physical, bodily pain, social functioning, general health perception, role emotional, and mental health) were also significantly more improved in the AKA-H group than in the sham group (all, P < 0.001). The scores for the physical, psychological, and social aspects of the SF-36 subscales showed similar improvement in the AKA-H group.

Conclusion

The AKA-H method can be effective in managing chronic low back pain.

Trial Registration

UMIN Clinical Trials Registry (UMIN-CTR) UMIN000006250.