Expression and characterization of recombinant C-terminal biotinylated extracellular domain of human receptor for advanced glycation end products (hsRAGE) in Escherichia coli

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K(d)) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.     

Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.     

Cytogenetic study of the induction mechanism of chromosome-type aberrations by 1-beta-D-arabinofuranosylcytosine

The bacteriophage P1 hot gene product is a homolog of the theta subunit of E. coli DNA polymerase III. Previous studies with hot cloned on a plasmid have shown that Hot protein can substitute for theta, as evidenced by its stabilizing effect on certain dnaQ mutator mutants carrying an unstable pol III proofreading subunit (varepsilon subunit). These results are consistent with Hot, like theta, being a replication protein involved in stabilizing the intrinsically unstable varepsilon proofreading function. However, the function of hot for the viral life cycle is less clear. In the present study, we show that the hot gene is not essential. Based on its promoter structure, hot has been previously classified as a "late" phage gene, a property that is not easily reconciled with a presumed replication function. Here, we clarify this issue by demonstrating that P1 hot is actively expressed both during the lysogenic state and in the early stages of a lytic induction, in addition to its expression in the late stage of phage development. The results indicate that P1 hot has a complex expression pattern, compatible with a model in which Hot may affect the host replication machinery to benefit overall phage replication.     

An Assessment of Integrated Risk Assessment

In order to promote international understanding and acceptance of the integrated risk assessment process, the World Health Organization/International Programme on Chemical Safety (WHO/IPCS), in collaboration with the U.S. Environmental Protection Agency and the Organization for Economic Cooperation and Development, initiated a number of activities related to integrated risk assessment. In this project, the WHO/IPCS defines integrated risk assessment as a science-based approach that combines the processes of risk estimation for humans, biota, and natural resources in one assessment. This article explores the strengths and weaknesses of integration as identified up to this date and the degree of acceptance of this concept by the global risk assessment/risk management community. It discusses both opportunities and impediments for further development and implementation.

The major emerging opportunities for an integrated approach stem from the increasing societal and political pressure to move away from vertebrate testing leading to a demand for scientific integrated approaches to in vitro and in vivo testing, as well as to computer simulations, in so-called Intelligent Testing Strategies. In addition, by weighing the evidence from conventional mammalian toxicology, ecotoxicology, human epidemiology, and eco-epidemiology, risk assessors could better characterize mechanisms of action and the forms of the relationships of exposures to responses. It is concluded that further demonstrations of scientific, economic and regulatory benefits of an integrated approach are needed. As risk assessment is becoming more mechanistic and molecular this may create an integrated approach based on common mechanisms and a common systems-biology approach.     

Poor compensatory function for sleep loss in delayed sleep phase syndrome and non-24-hour sleep-wake syndrome

Sleep and Biological Rhythms -