Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Diet of Japanese eels Anguilla japonica in the Kojima Bay-Asahi River system, Japan

The diet of Japanese eels, Anguilla japonica, was investigated using stomach content and stable isotope analyses. Stable isotope enrichment of carbon and nitrogen (Δδ¹³C and Δδ¹⁵N) was first estimated for A. japonica by comparing the isotopic signatures (δ¹³C and δ¹⁵N) of reared eels to that of their food. The estimated isotope enrichment was then applied to the diet estimation of A. japonica in the Kojima Bay-Asahi River system, Japan, combined with conventional stomach content analysis. Stable isotope enrichment varied among tissues, from 0.2‰ to 0.8‰ for carbon and from 1.3‰ to 2.1‰ for nitrogen. Nitrogen isotope enrichment of A. japonica muscle estimated in this study was 2.1‰, which was different from the previously reported mean δ¹⁵N enrichment of several animals of 3.4‰. These results indicate that isotope-based diet estimations for A. japonica need to use species- and tissue-specific values of isotope enrichment. In the diet analysis, stomach contents and stable isotopes revealed that (1) A. japonica appear to usually feed on a single type of prey species in each feeding session, (2) principal prey species were mud shrimp, Upogebia major, in brackish Kojima Bay and crayfish, Procambarus clarkia, in the Asahi River, (3) A. japonica in Kojima Bay primarily depend on the pelagic food web as a carbon source due to mud shrimp being filter feeders and eels in the Asahi River primarily depend on the littoral food web. Based on these results and the recently reported eel movements between Kojima Bay and the Asahi River, it appears that A. japonica can adapt to various feeding environments as opportunists, but also utilize the food resources by targeting a single type of prey species during a single feeding session.     

The Spherical Expansion of H2 Dimer Interaction Energy by Double Symmetry-Adapted Perturbation Theory

Abstract

The weak interaction energy of H₂ dimer is studied by double symmetry-adapted perturbation theory (SAPT) within second-order of intermolecular and intramonomer perturbation for molecular simulations. The assumed orientations of H₂ dimer are linear, parallel, T type and X type. Among four orientations T orientation is the most stable, while linear orientation is the most repulsive. The second-order dispersion energy E _disp ⁽²⁾ is the most attractive contribution in all orientations. The interaction energy has the anisotropy, so we expressed our total interaction energy by the spherical expansion to compare with the experimental value. The isotropic interaction energy is about 85% of the experimental value.     

Molecular dynamics simulation of fluorination effect for solvation of trifluoromethylbenzoic acid isomers in supercritical carbon dioxide

A molecular dynamics (MD) simulation was applied to carbon dioxide+trifluoromethylbenzoic acid isomer and carbon dioxide+methylbenzoic acid isomer systems to investigate the interactions between carbon dioxide and the solutes. The pair correlation functions between the carbon dioxide and trifluoromethyl group or methyl group in the solutes were calculated to study the fluorination effect of solvation. As a result, it was found that the interactions between carbon dioxide and trifluoromethyl group in trifluoromethylbenzoic acid isomers were stronger than those between carbon dioxide and the methyl group in methylbenzoic acid isomers. The simulation results had the same tendency as the experimental solubility enhancements and coincided with the trend of the interaction parameters of the Peng-Robinson equation of state that were determined from the solubility data.     

The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine

Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE = 1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase β

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1–9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol β, which is a DNA repair-related pol. Compounds 3–6 strongly inhibited the growth of human colon carcinoma HCT116 p53^+/+ cells. The influence of compounds 1–9 on HCT116 p53^?/? cell growth was similar to that observed for HCT116 p53^+/+ cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol β and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol β activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53^?/? cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol β inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.