Supercoiled DNA; plectonemic structure and liquid crystal formation

We have investigated the phase behaviour of pUC18 plasmid solutions with phase separation experiments and polarized light microscopy. Furthermore, the configuration of the superhelix is monitored with small-angle neutron scattering. The phase diagram is interpreted with liquid crystal theory including the effects of charge, orientation entropy, excluded volume, as well as the elastic, entropic and electrostatic contributions to the molecular free energy

Characterization of cell-induced astigmatism in high-resolution imaging

Supramolecular & Biomaterials Chemistr

In polycistronic Qβ RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

In Qβ RNA, sequestering the coat gene ribosome binding site in a putatively strong hairpin stem structure eliminated synthesis of coat protein and activated protein synthesis from the much weaker maturation gene initiation site, located 1300 nucleotides upstream. As the stability of a hairpin stem comprising the coat gene Shine–Dalgarno site was incrementally increased, there was a corresponding increase in translation of maturation protein. The effect of the downstream coat gene ribosome binding sequence on maturation gene expression appeared to have occurred only in cis and did not require an AUG start codon or initiation of coat protein synthesis. In all cases, no structural reorganization was predicted to occur within Qβ RNA. Our results suggest that protein synthesis from a relatively weak translational initiation site is greatly influenced by the presence or absence of a stronger ribosome binding site located elsewhere on the same RNA molecule. The data are consistent with a mechanism in which multiple ribosome binding sites compete in cis for translational initiations as a means of regulating protein synthesis on a polycistronic messenger RNA

Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin

The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A–B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A–B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death

Regulation of keratinocyte proliferation and differentiation by all-trans-retinoic acid, 9-cis-retinoic acid and 1,25-dihydroxy vitamin D3

We studied the effect of all-trans retinoic acid (all-trans-RA), 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) on proliferation and differentiation of human keratinocytes cultured in a submerged culture system for up to 5 weeks and evaluated changes in cell morphology and in the expression of proliferation- and terminal differentiation-related genes on both the mRNA and the protein levels. Under control culture conditions, the expression of small proline-rich proteins (SPRR1 and SPRR2), involucrin, Ki67 and c-jun reached a maximum after 2 weeks in culture (1 week postconfluence) and then decreased as the tissue architecture of the cultures deteriorated. Upon simultaneous treatment with both retinoids and 1,25(OH)2D3 a culture was generated that remained stable for 4 weeks with at least eight living cell layers. Furthermore, this culture showed a pattern of SPRR2 and involucrin expression which closely resembled that of native epidermis, a maintained Ki67 expression and a strongly induced c-jun expression. Treatment with 1,25(OH)2D3 alone inhibited cell proliferation and stimulated cell differentiation resulting in acceleration of the differentiated phenotype and was accompanied by inhibition of c-jun and Ki67 expression and also, surprisingly, by inhibition of SPRR1, SPRR2 and involucrin expression. In contrast, treatment with all-trans-RA and/or 9-cis-RA induced a more proliferative phenotype with a prolonged lifespan as compared to control cultures. SPRR1 was weakly repressed, SPRR2 was strongly repressed, a delayed induction of involucrin occurred, and c-jun and Ki67 expression were maintained. These results show that modulation of the composition of the medium by the addition of various vitamins results in changes in the balance between keratinocyte proliferation and differentiation which correspond to changes in the expression of proliferation and differentiation markers and prolongation of the culture lifespan

Superhelical stress restrained in plasmid DNA during repair synthesis initiated by the UvrA, B and C proteins in vitro.

Purified UvrA, UvrB, UvrC, UvrD, PolA and Lig proteins from Escherichia coli have been used to assess the effect of nucleotide excision repair on the conformation of native negatively supercoiled plasmid DNA in an in vitro test system. The analysis of labeled reaction products on specific gel systems suggests that the Uvr excinuclease has the ability to restrain the superhelical stress in the template DNA during the repair process. This feature, observed in the case of the Uvr system is not found if the repair reaction is initiated by T4 endonuclease V or Micrococcus luteus UV endonuclease