Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

Isotonic biaxial loading of fibroblast-populated collagen gels: a versatile, low-cost system for the study of mechanobiology

 We developed a simple, versatile system for applying a range of biaxial loads to cell–matrix constructs for the study of mechanobiology. The system consists of porous polyethylene bars that are polymerized into a square fibroblast-populated gel and loaded by freely hanging weights attached to sutures routed through a custom loading rig. The cost to manufacture each mold/loading rig pair was less than US $250 and the expected life of the components is up to 10 years. Neonatal and adult cardiac fibroblasts contracted gels to a decreasing extent as external load was increased (P=0.003) and achieved contraction forces of up to 1.4 mN per million cells. Strain distributions were reasonably homogeneous in the central region of the gel (25% of gel area), but clearly nonhomogeneous outside that central region. The primary advantages of this system are simplicity, low cost, biaxial loading, and the ability to test for a dose–response effect of mechanical load. The current disadvantages are the inability to apply cycling loading and the inhomogeneities introduced by the use of rigid loading bars. Received: 15 November 2001 / Accepted: 30 January 2002     

GDI-1 phosphorylation switch at serine 96 induces RhoA activation and increased endothelial permeability

We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Calpha-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 --> Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.     

Biosimilars--global issues, national solutions

Biotechnology derived medicinal products are presently the best characterized biologicals with considerable production and clinical experience, and have revolutionized the treatment of some of the most difficult-to-treat diseases, prolonging and improving the quality of life and patient care. They are also currently one of the fastest growing segments of the pharmaceutical industry market. The critical challenge that the biopharmaceutical industry is facing is the expiry of patents for the first generation of biopharmaceuticals, mainly recombinant DNA derived products, such as interferons, growth hormone and erythropoetin. The question that immediately arose was how should such copies of the originator products be licensed, bearing in mind that they are highly complex biological molecules produced by equally complex biological production processes with their inherent problem of biological variability. Copying biologicals is much more complex than copying small molecules and the critical issue was how to handle the licensing of products if relying in part on data from an innovator product. Since 2004 there has been considerable international consultation on how to deal with biosimilars and biological copy products. This has led to a better understanding of the challenges in the regulatory evaluation of the quality, safety and efficacy of "biosimilars", to the exchange of information between regulators, as well as to the identification of key issues. The aim of this article is to provide a brief overview of the scientific and regulatory challenges faced in developing and evaluating similar biotherapeutic products for global use. It is intended as an introduction to the series of articles in this special issue of Biologicals devoted to similar biotherapeutic products.     

Evaluation of similar biotherapeutic products (SBPs): scientific principles and their implementation

The availability of biotherapeutic products to patients has a major impact on the success in treating many life-threatening and chronic diseases. These products are often derived by recombinant DNA technology and are expensive for the majority of patients who need them most. Increasing numbers of patents/data protection are now expiring and biologicals "similar" to the originators (innovative products) are coming to the market. This process is seen as a mechanism for increasing the access to biotherapeutic products which are very much needed for the treatment of chronic diseases worldwide. The emergence of Similar Biological Products (SBPs) has created numerous challenges in developing, licensing, and using these important products. From a public health perspective, the overall expectation is that similar products will be available at an affordable price and will increase patients' access to the therapy. In response to the requests for assistance in defining regulatory requirements for SBPs, the WHO Expert Committee on Biological Standardization (ECBS) adopted the new WHO Guidelines for evaluation of SBPs in October 2009. This article provides a brief account of the WHO initiative to assist its member states to establish national requirements for the regulatory oversight of SBPs. The aim of the article is to inform its readers of the current status of WHO Guidelines on the evaluation of similar biotherapeutic products and of the plan to strengthen national regulatory requirements to assure quality, safety and efficacy of similar biotherapeutic products at the global level.     

Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis.     

The in vivo genotoxicity of cisplatin,isoflurane and halothane evaluated by alkaline comet assay in Swiss albino mice

The aim of this study was to evaluate the genotoxicity of repeated exposure to isoflurane or halothane and compare it with the genotoxicity of repeated exposure to cisplatin. We also determined the genotoxicity of combined treatment with inhalation anaesthetics and cisplatin on peripheral blood leucocytes (PBL), brain, liver and kidney cells of mice. The mice were divided into six groups as follows: control, cisplatin, isoflurane, cisplatin–isoflurane, halothane and cisplatin–halothane, and were exposed respectively for three consecutive days. The mice were treated with cisplatin or exposed to inhalation anaesthetic; the combined groups were exposed to inhalation anaesthetic after treatment with cisplatin. The alkaline comet assay was performed. All drugs had a strong genotoxicity (P < 0.05 vs. control group) in all of the observed cells. Isoflurane caused stronger DNA damage on the PBL and kidney cells, in contrast to halothane, which had stronger genotoxicity on brain and liver cells. The combination of cisplatin and isoflurane induced lower genotoxicity on PBL than isoflurane alone (P < 0.05). Halothane had the strongest effect on brain cells, but in the combined treatment with cisplatin, the effect decreased to the level of cisplatin alone. Halothane also induced the strongest DNA damage of the liver cells, while the combination with cisplatin increased its genotoxicity even more. The genotoxicity of cisplatin and isoflurane on kidney cells were nearly at the same level, but halothane caused a significantly lower effect. The combinations of inhalation anaesthetics with cisplatin had stronger effects on kidney cells than inhalation anaesthetics alone. The observed drugs and their combinations induced strong genotoxicity on all of the mentioned cells.     

Phages of Pseudomonas aeruginosa: response to environmental factors and in vitro ability to inhibit bacterial growth and biofilm formation

Aims: To examine effects of various environmental factors on adsorption and inactivation of Pseudomonas aeruginosa‐specific phages: δ (family Podoviridae), J‐1, σ‐1 and 001A (family Siphoviridae) and their ability to inhibit bacterial growth and biofilm formation. Methods and Results: The phages examined in the study were clonally different, as revealed by RFLP. The temperature in the range 7–44°C had no influence on the adsorption of Podoviridae, but did affect Siphoviridae adsorption, particularly 001A. All phages were significantly stable at pH 5–9, and phages δ and 001A even at pH 3. Most of the examined carbohydrates and exopolysaccharides of the original host efficiently inactivated phage δ, while phages σ‐1 and J‐1 were inactivated considerably only by the amino acid alanine. Silver nitrate efficiently inactivated all the phages, while Siphoviridae were more resistant to povidone‐iodine. Serum of nonimmunized rats had no influence on phage inactivation and adsorption. Only phage δ showed ability to effectively inhibit in vitro bacterial growth and biofilm formation. Conclusions: The examined environmental parameters can significantly influence the adsorption and viability of Ps. aeruginosa‐specific phages. The phage δ is a good candidate for biocontrol of Ps. aeruginosa. Significance and Impact of the Study: The study provides important data on Ps. aeruginosa‐specific phage adsorption, inactivation and in vitro lytic efficacy.