Preirradiation of host cells does not alter blockage of simian virus 40 replication forks by pyrimidine dimers

Do damage-inducible responses in mammalian cells alter the interaction of lesions with replication forks? We have previously demonstrated that preirradiation of the host cell mitigates UV inhibition of SV40 DNA replication; this mitigation can be detected within the first 30 min after the test irradiation. Here we test the hypotheses that this mitigation involves either (1) rapid dimer removal, (2) rapid synthesis of daughter strands past lesions (trans-dimer synthesis), or (3) continued progression of the replication fork beyond a dimer. Cells preirradiated with UV were infected with undamaged SV40, and the effects of UV upon viral DNA synthesis were measured within the first hour after a subsequent test irradiation. In preirradiated cells, as well as in non-preirradiated cells, pyrimidine dimers block elongation of daughter strands; daughter strands grow only to a size equal to the interdimer distance along the parental strands. There is, within this first hour after UV, no evidence for trans-dimer synthesis, nor for more rapid dimer removal either in the bulk of the parental DNA or in molecules in the replication pool. Progression of the replication forks was analyzed by electron microscopy of replicating SV40 molecules. Dimers block replication-fork progression in preirradiated cells to the same extent as in non-preirradiated cells. These experiments argue strongly against the hypotheses that preirradiation of host cells results in either the rapid removal of dimers, trans-dimer synthesis, or continued replication-fork progression beyond dimers.     

Novel role for E4 region genes in protection of adenovirus vectors from lysis by cytotoxic T lymphocytes

Target cells infected with adenovirus (Ad) vectors containing intact E3 and E4 regions were found to be relatively resistant to lysis by Ad-specific cytotoxic T lymphocytes. Elements from both the E3 and the E4 regions were required for this effect, leading to the identification of a previously undescribed role for E4 gene products in resistance to cytolysis.     

Mutations within the ADP (E3-11.6K) protein alter processing and localization of ADP and the kinetics of cell lysis of adenovirus-infected cells

ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic N(endo)C(exo) nuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2 adp gene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.     

New cationic lipids form channel-like pores in phospholipid bilayers

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.     

Comparative genomic and phenomic analysis of Clostridium difficile and Clostridium sordellii,two related pathogens with differing host tissue preference

Background

Clostridium difficile and C. sordellii are two anaerobic, spore forming, gram positive pathogens with a broad host range and the ability to cause lethal infections. Despite strong similarities between the two Clostridial strains, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues.

Results

In this study, to improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we have performed a comparative genomic and phenomic analysis of the two. The global phenomes of C. difficile and C. sordellii were compared using Biolog Phenotype microarrays. When compared to C. difficile, C. sordellii was found to better utilize more complex sources of carbon and nitrogen, including peptides. Phenotype microarray comparison also revealed that C. sordellii was better able to grow in acidic pH conditions. Using next generation sequencing technology, we determined the draft genome of C. sordellii strain 8483 and performed comparative genome analysis with C. difficile and other Clostridial genomes. Comparative genome analysis revealed the presence of several enzymes, including the urease gene cluster, specific to the C. sordellii genome that confer the ability of expanded peptide utilization and survival in acidic pH.

Conclusions

The identified phenotypes of C. sordellii might be important in causing wound and vaginal infections respectively. Proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies aimed at understanding C. difficile and C. sordellii infection site specificity and pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1663-5) contains supplementary material, which is available to authorized users.     

Helix → β conformational transition of poly(L-lysine) on dye binding

Binding of an azo dye, 4′-dimethyl amino azo benzene-4-carboxylic acid (DAAC) to poly(L -lysine) (PLL) in basic aqueous solutions at 20°C has been studied. The azo dye was found to bind to PLL when its side-chain amino groups are in the uncharged state. This was found to be a cooperative phenomenon, and the binding constant and cooperativity factor have been evaluated. The binding of the dye was found to result in a conformational transition of PLL from the α-helix to the β-sheet, which in turn helps in increased dye binding.     

Highly branched HK peptides are effective carriers of siRNA

BACKGROUND: Both viral and nonviral carriers have been used to carry small interfering RNA molecules (siRNA) to their cytosolic mRNA target. To date, few peptide carriers have been developed that have proved effective for siRNA delivery. Our previous branched carriers composed of histidine and lysine were useful for transfection of plasmids. In this study, we determined if these and more highly branched HK polymers were effective carriers of siRNA. METHODS: Several branched polymers were synthesized on a Ranin Voyager synthesizer. These polymers were then screened for their ability to transfer siRNA into SVR-bag4 cells, MDA-MB-435 cells, and C6 cells. After one polymer, H3K8b, was identified as an effective carrier of siRNA, additional polymers were synthesized to determine the essential domains for siRNA transport. The size/zeta-potential of HK : siRNA complexes were measured with the N4 submicron particle size analyzer and the Delsa 440 SX zeta-potential analyzer, respectively. Toxicity of the highly branched polymers in complex with siRNA was investigated by flow cytometry. RESULTS: In an endothelial cell line (SVR-bag4) that stably expressed beta-galactosidase (beta-gal), an siRNA in complex with the H3K8b polymer inhibited beta-gal expression by more than 80%. In contrast, the polymer H2K4b, which was an effective carrier of plasmids, was not an efficient carrier of siRNA. The size and surface charge did not distinguish effective from ineffective HK carriers of siRNA. By modifying H3K8b, we then determined what properties of H3K8b augmented siRNA delivery. The histidine-rich domain and the length of the terminal arms of H3K8 were important for siRNA delivery. The modestly more effective analog of H3K8b containing an integrin ligand, H3K8b(+RGD), was able to inhibit markedly intracellular beta-gal expression. Furthermore, we determined that H3K8b(+RGD) in complex with a luciferase-targeting siRNA inhibited luciferase expression in MDA-MB-435 cells. At its optimal concentration for inhibiting its target, H3K8b(+RGD) : siRNA complex had minimal toxicity. In contrast, carriers of siRNA such as Oligofectamine and Lipofectamine 2000 were significantly more toxic. CONCLUSIONS: Both the degree of complexity and the sequence specificity are important factors to be considered for developing the HK carrier of siRNA. In particular, we found that certain branched HK polymers (H3K8b, H3K8b(+RGD), and similar structural analogs) with eight terminal branches and a histidine-rich domain were effective carriers of siRNA.     

Sphingosine-1-phosphate agonists increase macrophage homing, lymphocyte contacts, and endothelial junctional complex formation in murine lymph nodes

The sphingosine-1-phosphate (S1P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S1P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S1P receptor 1 (S1P1)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S1P1-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S1P1, and treatment with FTY-P resulted in intense staining and colocalization of CD31, beta-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.