Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA and MxA-TAg NLS strongly interfered with the influenza virus transcription. Over-expression of PB2 led to a slight resumption of the transcription inhibition by nuclear MxA, whereas over-expression of PB1 and PA did not affect the MxA activity. Of interest is that the inhibitory activity of the nuclear MxA was markedly neutralized by over-expression of NP. An NP devoid of its C-terminal region, but containing the N-terminal RNA binding domain, also neutralized the VP16-MxA activity in a dose-dependent manner, whereas an NP lacking the N-terminal region did not affect the VP16-MxA activity. Further, not only VP16-MxA but also the wild-type MxA was found to interact with NP in influenza virus-infected cells. This indicates that the nuclear MxA suppresses the influenza virus transcription by interacting with not only PB2 but also NP.

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ABSTRACT. Inactivation of the klotho gene in mice causes serious systemic disorders, resembling human aging. However, at the molecular level, its action mechanisms are not well understood. The stimulatory or inhibitory effects of cis-and trans-regulatory factors on the klotho gene expression are also still unclear. We studied the effects of intra-and extracellular factors on human klotho gene expression. For this purpose, pHKP-Luc and pHKP-GFP reporter vectors were constructed with the 2.1-kbp upstream region of human klotho, covering its promoter region, using luciferase and GFP genes as the reporter. A series of vectors that have deletions in the upstream region of the klotho gene were constructed to assay cisacting factors. Deletion of some parts of the klotho gene upstream region significantly affected reporter gene expression in HEK293 cells. p16 and p53 proteins inhibited reporter luciferase expression under the control of human klotho promoter in a dose-dependent manner. Calcium and phosphate ions stimulated klotho expression. K. Turan and P. Ata p21, PTH, IGF-1, and angiotensin-II had no significant effect on klotho expression in HEK293 cells.

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Commercial chitosan (Ch) with low (LMWCh) and medium molecular weight (MMWCh) were hydrolyzed in diluted hydrochloric acid by heating at different temperatures. The viscosity average molecular weight of Chs was gradually decreased from 450 to 14 kDa as a function of temperature. Ch fractions were used for formation of Ch-DNA nanoparticles and tested for the ability to introduce DNA into HEK293, Swiss3T3, HeLa, and MDCK cells in vitro. The average diameter of nanoparticles was 200-220 nm. The surface charge of nanoparticles varied depending on the Ch/DNA ratio. The cell lines different response to DNA transfection with Ch fractions depended on molecular weight. HEK293 cells were efficiently transfected by nanoparticles prepared with Chs having a wide range of molecular weight (approximately 14-195 kDa). Swiss3T3 cells were efficiently transfected by Ch polymers with about <17 kDa. In contrast, HeLa and MDCK cells were highly resistant to DNA transfection with Ch polymers. These results strongly suggest that Ch polymers may be widely used for DNA trasnfection of the mammalian cells under optimized conditions.

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This paper describes the formulation of archaeosomes and the evaluation of their abilities to facilitate in vitro DNA delivery. Lipids of the H.hispanica 2TK2 strain were used in archaeosome formation, which is formulated by mixing H.hispanica 2TK2 lipids with plasmid DNA encoding green fluorescent protein (GFP) or β-galactosidase (β-gal). Archaeosome/pDNA formation and unbound DNA were monitored by agarose gel electrophoresis. The archaeosome formulations were visualized by AFM and TEM. The zeta potential analysis showed the archaeosomes to be electronegative. The composition of archaeosomes and the DNA dose for transient transfection into HEK293 cells were optimized, and the relationship between the structure and activity of archaeosomes in DNA delivery was investigated. By themselves, archaeosomes showed low efficiency for DNA delivery, due to their anionic nature. By formulating archaeosomes with a helper molecule, such as DOTAP, CaCl2, or LiCl, the capability of archaeosomes for gene transfection is significantly enhanced. The transfection profiles of efficient archaeosomes are proved to have a long shelf-life when maintained at room temperature. Thus, the archaeal lipids have the potential to be used as transfection reagents in vitro.

Tetra-coordinated nickel(II) complexes of two ONS (1, 2) and seven ONN (3a-3g) chelating 2-hydroxy-3-methoxy-benzaldehyde thiosemicarbazones were synthesized. The dibasic ligands and complexes bearing PPh 3 as a coligand were characterized by means of analytical and spectroscopic data. Cytotoxic activities of the ligands and nickel(II) complexes were determined using the MTT assay in vitro against MDCK cells, and then all the compounds were tested on influenza virus replication by plaque assays. The compounds showed GI 50 values varying from concentrations of 15.9 up to 161.8 µ g/mL for MDCK cells. The plaque assays indicated that one ONS (1) and two ONN (3c and 3d) chelate structures have considerable antiviral effects on influenza A viruses at lower concentrations than the GI 50 values for MDCK cells. The ligands and other complexes did not show any inhibitory effects on influenza virus plaque formation. The effects of the compounds on the influenza virus and structure-antiviral activity relationships were discussed based on the donor atoms and S-alkyl substituents.