Treatment of whole blood (WB) and red blood cells (RBC) with S-303 inactivates pathogens and retains in vitro quality of stored RBC.

A pathogen inactivation (PI) process has been developed using the frangible anchor linker effector (FRALE) compound S-303. A series of experiments were performed in whole blood (WB) to measure the level of viral and bacterial inactivation. The results showed that 0.2mM S-303 and 2mM glutathione (GSH) inactivated >6.5 logs of HIV, >5.7 logs of Bluetongue virus, >7.0 logs of Yersinia enterocolitica, 4.2 logs of Serratia marcescens, and 7.5 logs of Staphylococcus epidermidis. Recent development for S-303 is focused on optimization of the PI process for red blood cell concentrates (RBC). A series of studies in RBC showed that 0.2mM S-303 and 20mM GSH inactivated approximately 5 logs or greater of Y. enterocolitica, E. coli, S. marcescens, S. aureus, HIV, bovine viral diarrhoea virus, bluetongue virus and human adenovirus 5. In both applications of the S-303 process, in vitro parameters of RBC function and physiology were retained compared to conventional RBC. Results from these studies indicate that S-303 can be applicable for PI of RBC and WB.

Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex.

Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selective filtration processes; sequestration of growth factors; and regulation of cellular differentiation. Furthermore, expression PGs has been found to vary in several disease states. In order to elucidate the role of PGs in the BM, a well-characterized model of polarized epithelium, Madin-Darby canine kidney (MDCK) cells has been utilized. Proteoglycans were prepared from conditioned medium by DEAE anion exchange chromatography. The eluted PGs were treated with heparitinase or chondroitinase ABC (cABC), separately or combined, followed by SDS-PAGE. Western blot analysis, using antibodies specific for various PG core proteins or CS stubs generated by cABC treatment, revealed that both basement membrane and interstitial PGs are secreted by MDCK cells. HSPGs expressed by MDCK cells are perlecan, agrin, and collagen XVIII. Various CSPG core proteins are made by MDCK cells and have been identified as biglycan, bamacan, and versican (PG-M). These PGs are also associated with mammalian kidney tubules in vivo.

Still more complexity in mammalian basement membranes.

At the epithelial/mesenchymal interface of most tissues lies the basement membrane (BM). These thin sheets of highly specialized extracellular matrix vary in composition in a tissue-specific manner, and during development and repair. For about two decades it has been apparent that all BMs contain laminins, entactin-1/nidogen-1, Type IV collagen, and proteoglycans. However, within the past few years this complexity has increased as new components are described. The entactin/nidogen (E/N) family has expanded with the recent description of a new isoform, E/N-2/osteonidogen. Agrin and Type XVIII collagen have been reclassified as heparan sulfate proteoglycans (HSPGs), expanding the repertoire of HSPGs in the BM. The laminin family has become more diverse as new alpha-chains have been characterized, increasing the number of laminin isoforms. Interactions between BM components are now appreciated to be regulated through multiple, mostly domain-specific mechanisms. Understanding the functions of individual BM components and their assembly into macromolecular complexes is a considerable challenge that may increase as further BM and cell surface ligands are discovered for these proteins.

Tau self-association: stabilization with a chemical cross-linker and modulation by phosphorylation and oxidation state.

tau is a major component of paired helical filaments found in the neurofibrillary tangles of Alzheimer's diseased brain. However, the mechanism or mechanisms responsible for the association of tau to form these aggregates remains unknown. In this study, the role of intermolecular disulfide bonds in the formation of higher order oligomers of bovine tau and the human recombinant tau isoform T3 was examined using the chemical cross-linking agent disuccinimidylsuberate (DSS). In addition, the role of phosphorylation and oxidation state on the in vitro self-association of tau was studied using this experimental model. Stabilization of tau-tau interactions with DSS indicated that intermolecular disulfide bonds probably play a predominant role in dimer formation, but the formation of higher order oligomers of tau cannot be attributed to these bonds alone. tau-tau interactions were significantly decreased either by blocking Cys residues or by exposing the tau to a reducing (nitrogen and dithiothreitol), instead of an oxidizing, environment. tau self-association was also significantly decreased by prior phosphorylation with calcium/calmodulin-dependent protein kinase II. Phosphorylation by cyclic AMP-dependent protein kinase or dephosphorylation by alkaline phosphatase did not alter tau self-assembly. These data suggest a role for several factors that may modulate tau self-association in vivo.

Metal (Fe3+) affinity chromatography: differential adsorption of tau phosphoproteins.

Tau is a neuronal cytoskeletal protein consisting of a group of isoforms with apparent molecular masses ranging from 45 to 62 kDa. Tau purified from brain exists in multiple phosphorylated forms and abnormally phosphorylated tau appears to play an important role in the neuropathology of Alzheimer's disease. To separate the differentially phosphorylated populations of tau, a chromatographic technique using ferric ions adsorbed onto iminodiacetic acid substituted Sepharose was developed. Several distinct populations of tau were isolated based on the phosphorylation state. These preparations can be used for further investigation of how each specific phosphorylation state modulates the metabolism and function of tau.

Past lessons and new uses of the mass media in reducing tobacco consumption.

A review of mass media response to the smoking issue over the past 25 years reveals that sustained involvement of the broadcast and print media has served significantly to heighten public awareness and reduce smoking rates in the total U.S. population. Public service advertising has been an integral part of the smoking control movement from its outset, but today's intensely competitive media environment has forced health promoters to look beyond public service announcements in the development of total communication programs. Media advocacy--using the media to sharpen public awareness and mold public policy to serve the public interest, a technique derived from political campaigns--is emerging as a powerful tool in the smoking control movement. Its emphasis is on changing the entire social context of tobacco use in America, rather than the smoking behavior of people. Because media advocates' success pivots on their access to the media, they must be able both to create news and to react quickly to breaking news and unexpected events. The opportunistic, risk-taking nature of media advocacy requires that most efforts be waged at the State and local levels. An increasing number of State health departments and other organizations are using paid advertising to improve the frequency and reach of nonsmoking messages. Research verifies that paid media campaigns increase the target audience's exposure to smoking control messages, but planning and making efficient media purchases require sophistication and, of course, the necessary funds. Irrefutable medical evidence linking smoking to disease and addiction, combined with the powerful social force of the nonsmokers' rights movement, offer hope that a smoke-free society is an achievable goal. Success,however, will only be realized if tobacco control activists make use of the full range of mass media technologies to sustain and nourish this momentum.