Inorganic polyphosphate interacts with ribosomes and promotes translation fidelity in vitro and in vivo

Inorganic polyphosphate is a biological macromolecule consisting of multiple phosphates linked by high-energy bonds. Polyphosphate occurs in cells from all domains of life, and is known to play roles in a diverse collection of cellular functions. Here we examine the relationship between polyphosphate and protein synthesis in Escherichia coli. We report that polyphosphate associates with E. coli ribosomes in vitro. Characterization of this interaction reveals that both long-chain and short-chain polyphosphates interact with the ribosome. Intact 70S ribosomes, as well as 50S and 30S subunits, display a specific interaction with polyphosphate that is mediated primarily by contacts with ribosomal proteins. Additionally, we examined functional consequences of a ppk mutation, which severely reduces levels of intracellular polyphosphate. Extracts from ppk mutants contain lower levels of polysomes than wild-type cells, suggesting a defect in mRNA utilization or the mRNA-ribosome interaction. Ribosomes from wild-type and ppk mutant cells were isolated, and their activities were compared using a polyU RNA in vitro translation assay. While rates of polyphenylalanine synthesis are similar, use of ribosomes from ppk cells results in a misincorporation rate about five times higher compared with the rate observed when ribosomes from wild-type cells are used. Mistranslation rates in vivo were measured directly, and ppk mutants displayed higher readthrough frequencies for two different stop codons. Taken together, these results indicate that polyphosphate plays an important role in maintaining optimal translation efficiency in vivo and in vitro.     

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming

Induced pluripotent stem cells (iPSCs) are considered patient‐specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c‐Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical‐grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non‐integrating viral and non‐viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.     

In vitro and in vivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.     

On the fidelity of DNA replication: manganese mutagenesis in vitro

Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates. Using Escherichia coli DNA polymerase I with poly[d(A-T)] and phi X174 DNA templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates. This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated at high fidelity. Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection. (2) At low free Mn2+ concentrations (less than 100 microM), manganese causes errors in incorporation via its interaction with the DNA template. The concentration dependence of mutagenesis is determined by the strength of binding Mn2+ to the particular DNA template used. The data do not allow one to rule out the possibility that Mn2+-deoxynucleoside triphosphate interactions contribute to mutagenesis in selected situations. This range of free Mn2+ concentrations is the one of greatest relevance for in vivo mutagenesis. (3) At higher concentrations (between 500 microM and 1.5 mM), further mutagenesis by Mn2+ occurs. This mutagenesis probably is due either to binding of manganese to single-stranded regions within the DNA or to weak accessory sites on the enzyme.     

Antiviral activities of resveratrol against rotavirus in vitro and in vivo

BackgroundRotavirus (RV) is the primary causative agent for viral gastroenteritis among infants and young children worldwide. Currently, no clinically approved and effective antiviral drug for the treatment of RV infection is available.PurposeWe investigated the potential anti-RV activity of resveratrol and underlying mechanisms by which resveratrol acted against RV.MethodsThe anti-RV activity of resveratrol in vitro was evaluated using plaque reduction assays. The effects of resveratrol on yield of virion progeny, viral polyprotein expression and genomic RNA synthesis were respectively investigated using enzyme-linked immunosorbent assays, western blotting and qRT-PCR assays. Further, we also measured the antiviral effect of resveratrol by evaluation of antigen clearance and assessment of changes in proinflammatory cytokines/chemokines in RV-infected neonatal mouse model.ResultsOur results indicated that 20 μM of resveratrol significantly inhibited RV replication in Caco-2 cell line by suppressing RV RNA synthesis, protein expression, viroplasm plaque formation, progeny virion production, and RV-induced cytopathy independent of the different strains and cell lines of RV that we used. Analysis of the effect of time post-addition of resveratrol indicated that its application inhibited early processes in the RV replication cycle. Further study of the underlying mechanism of anti-RV activity indicated that resveratrol inhibited RV replication by suppressing expression of heat-shock protein 90 (HSP90) mRNA and protein, and that the effect occurred in a dose-dependent manner. Overexpression of HSP90 was found to have attenuated the inhibitory effect of resveratrol on RV replication. Interestingly, the application of resveratrol were found to down-regulate the level of inhibition of RV-mediated MEK1/2 and ERK phosphorylation. Using a RV-infected suckling mice model, we found that application of resveratrol significantly lessened the severity of diarrhea, decreased viral titers, and relieved associated symptoms. Levels of mRNA expression of interleukin-2, interleukin-10, tumor necrosis factor-α, interferon-γ, macrophage inflammatory protein 1, and monocyte chemotactic protein-1 were all found to have been sharply reduced in intestinal tissue from mice which had been treated with resveratrol (10 or 20 mg/kg) after RV infection (p < 0.05).ConclusionThese findings implied that resveratrol exhibits antiviral activity and could be a promising treatment for rotavirus infection.