Chemical synthesis of the T4 endonuclease V gene and its expression in Escherichia coli

A gene coding for T4 endonuclease V was constructed by joining chemically-synthesized oligodeoxyribonucleotides. This gene was inserted into an expression vector and expressed in E. coli.     

A quartz crystal viscosity sensor for monitoring coagulation reaction and its application to a multichannel coagulation detector

A quartz crystal viscosity sensor was applied to a coagulation reaction monitoring system. The system consists of 16 oscillating circuits, a channel selector, a frequency counter, a temperature controller and a microcomputer. The system is named the Quartz Chemical Analyzer (QCA). AT-cut quartz crystals (9 MHz) were used as viscosity detectors and were attached to a cell in order to expose only one side of the quartz plate. The system was applied to the detection of the blood coagulation factors VIII (F VIII) and IX (F IX). The activity of these factors was assayed by a single-stage method. A linear relationship was obtained in a double-logarithmic diagram of concentration versus coagulation time with respect to F VIII and F IX in the range 0.05-0.4 unit cm-3 and 0.025-0.2 unit cm-3, respectively.     

A KALA-modified lipid nanoparticle containing CpG-free plasmid DNA as a potential DNA vaccine carrier for antigen presentation and as an immune-stimulative adjuvant

Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms α-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-β and IL-1β production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine.     

Stability difference between DNA and RNA mini-hairpins containing two G-C pairs.

The deoxyribonucleotide fragments, d(GCGAAAGC) and d(GCGAAGC) form extraordinarily stable mini-hairpin structures containing only two G-C pairs. In contrast, the corresponding ribonucleotide fragments, r(GCGAAAGC) and r(GCGAAGC) are not so stable, although they also form mini-hairpin structures. The stability difference between these DNA and RNA mini-hairpins was examined by NMR studies and the molecular mechanics calculations, and by comparing with the stability of several sequence variants. Their stability was deduced to vary delicately depending on helical patterns formed by DNA (B form) and RNA (A form) structures.     

In vivo visualization of oxyradical-dependent photoemission during endothelium-granulocyte interaction in microvascular beds treated with platelet-activating factor

Oxyradical-dependent chemiluminescence from granulocytes sticking to venular endothelium was successfully visualized in rat mesenteric microvascular beds treated with platelet-activating factor (PAF). Intravital photonic image intensifier microscopy revealed that topical application of PAF-acether (100 nM) caused remarkable granulocyte adherence on endothelial walls and the subsequent activation of a luminol-dependent photonic burst. Chemilumigenic sites clearly corresponded to the spatial distribution of sticking cells in post-capillary venules. The present findings thus serve as the first demonstration of an intravital oxidative burst of granulocytes on venular endothelium in PAF-induced microcirculatory disturbances.     

Serum thyroglobulin concentration in patients with diabetes mellitus

The serum thyroglobulin (Tg) concentration was measured in 97 patients with diabetes mellitus (39 males, 58 females). Hyper Tg-nemia which exceeds the normal range (1.0-26.6 ng/ml) was observed in 10 patients (3 out of 21 cases treated with diet alone, 3 out of 50 cases treated with oral hypoglycemic agents, 4 out of 26 cases treated with insulin). There was no significant correlation between concentrations of serum Tg and triiodothyronine (T3), thyroxine (T4), fasting plasma glucose (FPG), and hemoglobin A1c (HbA1c). However, a positive correlation was observed between serum concentrations of Tg and thyroid stimulating hormone (TSH). Patients with diabetes were divided into three groups according to the mode of treatment (Group I; diet alone, n = 21, Group II; oral hypoglycemic agents, n = 50, Group III; insulin, n = 26). No significant difference in the serum Tg concentration was observed among the three groups. They were also divided into two groups; normal Tg-nemia (Group A, n = 87) and hyper Tg-nemia (Group B, n = 10). There was no difference between levels of T3, T4, FPG, and HbA1c in the two groups. The serum TSH concentration measured by double antibody RIA and two site immunoradiometric assay in Group B was significantly higher than that in Group A. These results suggest that hyper Tg-nemia in patients with diabetes could be due to the increased TSH concentration which reflects latent subclinical primary hypothyroidism in them.     

Studies on the Metabolism of Fluoroacetate in the Liver of a Rat

In a rat poisoned with sodium fluoroacetate no accumulation of citrate was found in the liver, while in that of a frog fluoroacetate adtninistration was found to cause a marked increase of citrate. In this paper, it was shown that even in a rat when it was treated with insulin before fluoroacetate administration, citrate accuillulation could be found in the liver. In the experiments in vitro using slices of a rat liver it was also shown that fluoroacetate could cause an accumulation of citrate and inhibited the oxidation of malate in the slices of a normal as well as an insulinized liver.     

A Novel Polysaccharide in Insects Activates the Innate Immune System in Mouse Macrophage RAW264 Cells

A novel water-soluble polysaccharide was identified in the pupae of the melon fly (Bactrocera cucurbitae) as a molecule that activates the mammalian innate immune response. We attempted to purify this innate immune activator using nitric oxide (NO) production in mouse RAW264 macrophages as an indicator of immunostimulatory activity. A novel acidic polysaccharide was identified, which we named “dipterose”, with a molecular weight of 1.01×10⁶ and comprising nine monosaccharides. Dipterose was synthesized in the melon fly itself at the pupal stage. The NO-producing activity of dipterose was approximately equal to that of lipopolysaccharide, a potent immunostimulator. Inhibition of Toll-like receptor 4 (TLR4) led to the suppression of NO production by dipterose. Furthermore, dipterose induced the expression of proinflammatory cytokines and interferon β (IFNβ) and promoted the activation of nuclear factor kappa B (NF-κB) in macrophages, indicating that it stimulates the induction of various cytokines in RAW264 cells via the TLR4 signaling pathway. Our results thus suggest that dipterose activates the innate immune response against various pathogenic microorganisms and viral infections. This is the first identification of an innate immune-activating polysaccharide from an animal.