Targeted delivery to macrophages and dendritic cells by chemically modified mannose ligand-conjugated siRNA

Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.     

Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.     

Limited Internal Radiation Exposure Associated with Resettlements to a Radiation-Contaminated Homeland after the Fukushima Daiichi Nuclear Disaster

Resettlement to their radiation-contaminated hometown could be an option for people displaced at the time of a nuclear disaster; however, little information is available on the safety implications of these resettlement programs. Kawauchi village, located 12–30 km southwest of the Fukushima Daiichi nuclear power plant, was one of the 11 municipalities where mandatory evacuation was ordered by the central government. This village was also the first municipality to organize the return of the villagers. To assess the validity of the Kawauchi villagers’ resettlement program, the levels of internal Cesium (Cs) exposures were comparatively measured in returnees, commuters, and non-returnees among the Kawauchi villagers using a whole body counter. Of 149 individuals, 5 villagers had traceable levels of Cs exposure; the median detected level was 333 Bq/body (range, 309–1050 Bq/kg), and 5.3 Bq/kg (range, 5.1–18.2 Bq/kg). Median annual effective doses of villagers with traceable Cs were 1.1 x 10^-2 mSv/y (range, 1.0 x 10^-2-4.1 x 10^-2 mSv/y). Although returnees had higher chances of consuming locally produced vegetables, Cochran-Mantel-Haenszel test showed that their level of internal radiation exposure was not significantly higher than that in the other 2 groups (p=0.643). The present findings in Kawauchi village imply that it is possible to maintain internal radiation exposure at very low levels even in a highly radiation-contaminated region at the time of a nuclear disaster. Moreover, the risks for internal radiation exposure could be limited with a strict food control intervention after resettlement to the radiation-contaminated village. It is crucial to establish an adequate number of radio-contaminated testing sites within the village, to provide immediate test result feedback to the villagers, and to provide education regarding the importance of re-testing in reducing the risk of high internal radiation exposure.     

Transformation of Citric Acid to Acetic Acid,Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.

Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg²⁺ and Ag²⁺ . The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl₂, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malo-lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.     

SMAP2 Regulates Retrograde Transport from Recycling Endosomes to the Golgi

Retrograde transport is where proteins and lipids are transported back from the plasma membrane (PM) and endosomes to the Golgi, and crucial for a diverse range of cellular functions. Recycling endosomes (REs) serve as a sorting station for the retrograde transport and we recently identified evection-2, an RE protein with a pleckstrin homology (PH) domain, as an essential factor of this pathway. How evection-2 regulates retrograde transport from REs to the Golgi is not well understood. Here, we report that evection-2 binds to SMAP2, an Arf GTPase-activating protein. Endogenous SMAP2 localized mostly in REs and to a lesser extent, the trans-Golgi network (TGN). SMAP2 binds evection-2, and the RE localization of SMAP2 was abolished in cells depleted of evection-2. Knockdown of SMAP2, like that of evection-2, impaired the retrograde transport of cholera toxin B subunit (CTxB) from REs. These findings suggest that evection-2 recruits SMAP2 to REs, thereby regulating the retrograde transport of CTxB from REs to the Golgi.     

Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase

The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B–dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.     

The first epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Okinawa, Japan, in 1985-1986

Epidemics of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant occurred in July-November, 1985 and August-October, 1986 in Okinawa Prefecture, Japan. This is the first report of an acute hemorrhagic conjunctivitis epidemic due to a coxsackievirus A24 variant in Japan The epidemic involved most islands of the prefecture. The prefectural surveillance center was notified of 9,952 cases in 1985 and 6,096 cases in 1986 from three sentinel eye clinics. The neutralizing antibody-positive rate against the coxsackievirus A24 variant of the serum samples collected before and immediately after the 1985 epidemic rose from 1.0% to 8.5%. The coxsackievirus A24 variant was isolated from 48 out of 68 conjunctival swabs collected during the epidemics. The isolates were indistinguishable antigenically in the plaque reduction test from the prototype strain, EH24/70, but had a markedly distinct oligonucleotide pattern.     

Levallorphan and dynorphin improve motor dysfunction in Mongolian gerbils with unilateral carotid occlusion: the first application of the inclined plane method in the experimental cerebral ischemia

Levallorphan and dynorphin were effective on the motor dysfunction in the gerbil model of unilateral cerebral ischemia. The effect of opioids, levallorphan (mixed agonist-antagonist), dynorphin (kappa-receptor agonist) and naloxone (mu-receptor antagonist), on neurological impairment was evaluated using the unilateral cerebral ischemia model of gerbil. Motor function was evaluated quantitatively by using the inclined plane method. Both levallorphan-treated group and dynorphin-treated group showed a significant improvement of the motor dysfunction compared with saline-control group. On the other hand, naloxone-treated group did not differ from saline-control group. The beneficial effect of these opioids on motor dysfunction might be mediated by the kappa-opioid receptor. This study also showed the potential usefulness of the inclined plane method for the investigation on the cerebral ischemia.