Structures of the modified nucleoside analogs (), sequences and numbering schemes (), and thermal denaturation of the DNA:DNA and DNA:RNA duplexes ()

<p><b>Copyright information:</b></p><p>Taken from "Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules"</p><p></p><p>Nucleic Acids Research 2007;35(6):1969-1977.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874594.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The thermal denaturation of the duplexes was performed as described in ()

Final 2 − maps contoured at 1σ level for the base pairs: U8:A17 in DD1b () and U5:A14 in DR2a ()

<p><b>Copyright information:</b></p><p>Taken from "Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules"</p><p></p><p>Nucleic Acids Research 2007;35(6):1969-1977.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874594.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p

Intraduplex interactions involving the 20th residues in the DD1a (), DD1b () and DD2 () DNA:DNA duplexes, and the 5th residues in the DR2a (), DR2b () and DR2c () DNA:RNA duplexes

<p><b>Copyright information:</b></p><p>Taken from "Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules"</p><p></p><p>Nucleic Acids Research 2007;35(6):1969-1977.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874594.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The carbon atoms in the aminohexyl, carbamoyl and methoxyl modifications are colored green, and the water molecules are colored pale blue. Broken and dotted lines indicate possible hydrogen bonds and van der Waals interactions, respectively. The values indicated are in angstroms (Å)

Hydration structures in the minor grooves of the unmodified (), DD1a (), DD1b () and DD2 () DNA:DNA duplexes

<p><b>Copyright information:</b></p><p>Taken from "Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules"</p><p></p><p>Nucleic Acids Research 2007;35(6):1969-1977.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874594.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The unmodified duplex is shown in blue lines while the present duplexes are shown in red lines. The aminohexyl, carbamoyl and methoxyl groups are colored green. In the unmodified duplex, the cyan spheres are water molecules and the gray spheres are solvent molecules partially occupied by sodium ions and water molecules. In DD1a and DD1b, the water molecules are in cyan, and the potassium ions are in gray

The minor groove widths in the DD1a (open square), DD1b (○), DD2 (open triangle) and unmodified (multi) DNA:DNA duplexes, and in the DR2a(filled square), DR2b (filled circle), DR2c (filled triangle) and unmodified (asterisk) DNA:RNA hybrid duplexes

<p><b>Copyright information:</b></p><p>Taken from "Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules"</p><p></p><p>Nucleic Acids Research 2007;35(6):1969-1977.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874594.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The minor groove width is defined as the distance between the closest interstrand phosphates, diminished by 5.8 Å to account for the van der Waals radii of the phosphate groups (). X is thymine in the unmodified duplexes and U or U in the modified duplexes

Functional differentiation of CD4⁺ T cells is impaired in CD98hc^f/f-CD4 mice.

<p>CD98hc^f/f-CD4 or CD98hc^+/+-CD4 mice were immunized with OVA protein emulsified in CFA. (A) Serum anti-OVA specific IgG, IgG1, IgG2a, IgG2c, and IgM titers at eight days after OVA immunization were determined by ELISA. As negative controls, sera from unimmunized CD98hc^f/f-CD4 and CD98hc^+/+-CD4 mice were used. Results are means ± S.D. of 7 mice; * significant difference (<i>p</i><0.05). (B) Total lymph node cells from OVA-immunized CD98hc^f/f-CD4 or CD98hc^+/+-CD4 mice were stimulated with OVA protein for 3 days. [³H]-thymidine incorporation during the final 6 hours was determined. Results are means ± S.D. of 5 mice; * significant difference (<i>p</i><0.05). (C) After stimulating T cells from OVA immunized CD98hc^f/f-CD4 and CD98hc^+/+-CD4 mice with OVA protein (50 μg/ml) for 3 days, culture supernatant concentrations of IL–4, IL–17, and IFN-γ were determined by ELISA. Results are means ± S.D. of 3 mice; * significant difference (<i>p</i><0.05). Data shown in this Figure are representative of three experiments.</p

IFN-γ secretion is disturbed in CD98hc^f/f-CD4 mice.

<p>(A) CFSE labeled spleen cells from CD98hc^f/f-CD4-OT11 or CD98hc^+/+-CD4-OT11 mice were stimulated with OVA peptides under Th1 culture conditions for 3 days. Cells were stained with anti-CD4, anti-CD98hc and anti-IFN-γ mAbs and evaluated by flow cytometry. Data shown are gated on CD98hc^- cells. (B) Spleen cells from CD98hc^f/f-CD4-OT11 or CD98hc^+/+-CD4-OT11 mice were stimulated with OVA peptides under Th1 culture conditions for 3 days. Cells were stained with anti-CD4 and AnnexinV, and evaluated by flow cytometry. (C) Cells from CD98hc^f/f-CD4-OT11 (Thy1.2) or CD98hc^+/+-CD4-OT11 (Thy1.1⁺/Thy1.2⁺) mice were labeled with CFSE and transferred into CD45.1 C57BL/6 mice. Mice were then immunized with OVA protein. CFSE dilution and intracellular INF-γ expression were evaluated by flow cytometry gated on CD98hc⁺ (CD98hc^+/+-CD4-OT11) or CD98^- cells (CD98hc^f/f-CD4-OT11) cells four days after immunization (left). The percentages of INF-γ⁺ cells among total CD4⁺ T cells are shown (middle). The percentages of INF-γ⁺ cells among total CD4⁺ cells that underwent cell division at the indicated times were counted (right). Results are means ± S.D. of 4 mice; * significant difference (<i>p</i><0.05). Data shown in this Figure are representative of three experiments.</p

T cell development is normal in CD98hc^f/f-CD4 mice.

<p>(A) CD4⁺TCRβ⁺, CD8⁺TCRβ⁺, CD19⁺, and CD11c⁺ cells in the spleens and CD4⁺TCRβ⁺, CD8⁺TCRβ⁺, CD4^-CD8^- (DN) and CD4⁺CD8⁺ (DP) cells in the thymus of CD98hc^f/f-CD4 mice (solid line) and CD98hc^+/+-CD4 mice (black shadow) were stained with an anti-CD98hc mAb. CD98hc expression was evaluated by flow cytometry. Unstained cells were used as a negative control (gray shadow). (B) Thymocytes from CD98hc^f/f-CD4 (closed) and CD98hc^+/+-CD4 (open) mice were stained with anti-CD4 and anti-CD8 mAbs. Total cell numbers for CD4^-CD8^- (DN; double negative), CD4⁺CD8⁺ (DP; double positive), CD4⁺CD8^- (CD4⁺), or CD4^-CD8⁺ (CD8⁺) cells were evaluated (left). CD69 expression on CD4⁺CD8⁺, CD4⁺CD8^-, or CD4^-CD8⁺ cells was evaluated by flow cytometry (right). (C) Spleen cells (left) and lymph node cells (right) from CD98hc^f/f-CD4 (closed) and CD98hc^+/+-CD4 (open) mice were stained with anti-CD4, anti-CD8, anti-CD44, or anti-CD62L mAbs; total cells from 6 mice were counted. Results are means ± S.D. Data shown in this Figure are representative of three independent experiments.</p

T cells from CD98hc^f/f-CD4 mice cannot mount immune responses against-<i>Leishmania major</i>.

<p>CD98hc^+/+-CD4 or CD98hc^f/f-CD4 mice under a C57BL/6 background were infected with <i>Leishmania majo</i>r in the footpad. (A) Footpad swelling in CD98hc^+/+-CD4 (closed) or CD98hc^f/f-CD4 (open) mice was measured after infection. Results are means ± S.D. of 8 mice; *or ** significant difference (<i>p</i> < 0.05 or p < 0.01, respectively). (B) Total popliteal lymph nodes from <i>Leishmania major</i>-infected CD98hc^+/+-CD4 or CD98hc^f/f-CD4 mice (day 30) were cultured for 5 days. Then, parasite numbers were counted and parasites/lymph node cells were determined. Results are means ± S.D. of 8 mice; ** significant difference (<i>p</i><0.01). (C) Purified CD4⁺ T cells (5 x 10⁵/well) from lymph node cells from CD98hc^f/f-CD4 (closed) or CD98hc^+/+-CD4 (open) mice infected with <i>Leishmania major</i> (day 10 and day 70) were stimulated with irradiated spleen cells (2 x 10⁵/well) and parasite-derived antigens for 3 days. Then, IFN-γ and IL–4 in culture supernatants were determined by ELISA. Results are means ± S.D. of 8 mice; * or ** significant difference (<i>p</i><0.05 or p < 0.01, respectively). Purified CD4⁺ T cells (5 x 10⁵/well) from lymph node cells from CD98hc^f/f-CD4 (closed circle) or CD98hc^+/+-CD4 (open circle) mice infected with <i>Leishmania major</i> (day 20) were stimulated with irradiated spleen cells (2 x 10⁵/well) and parasite-derived antigens for 3 days. Then, [³H]-thymidine incorporation was determined. Results are means ± S.D. from 8 mice; ** significant difference (<i>p</i><0.01). Data shown in this Figure are representative of three experiments.</p

Host-derived apolipoproteins play comparable roles with viral secretory proteins E^rns and NS1 in the infectious particle formation of <i>Flaviviridae</i>

<div><p>Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the <i>Flaviviridae</i> members, pestivirus and flavivirus possess a viral structural protein E^rns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while <i>Hepacivirus</i> including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of E^rns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded E^rns and NS1 in the formation of infectious particles. We examined whether E^rns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either E^rns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an E^rns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of E^rns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of <i>Flaviviridae</i>, especially in the maturation of infectious particles, while E^rns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.</p></div