Interrogating TGF-ß Function and Regulation in Endothelial Cells.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine with important roles in embryogenesis and maintaining tissue homeostasis during adult life. There are three isoforms of TGF-ß, i.e., TGF-ß1, -ß2, and -ß3, which signal by binding to a complex of transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. In most cell types TGF-ß signals via TGF-ß type II receptor (TßRII) and TßRI, also termed activin receptor-like kinase 5 (ALK5). In endothelial cells, TGF-ß signals via ALK5 and ALK1. These two type I receptors mediate opposite cellular response for TGF-ß. The co-receptor endoglin, highly expressed on proliferating endothelial cells, facilitates TGF-ß/ALK1 and inhibits TGF-ß/ALK5 signaling. Knockout of TGF-ß receptors in mice all result in embryonic lethality during midgestation from defects in angiogenesis, illustrating the pivotal role of TGF-ß in this process. This chapter introduces methods for examining the function and regulation of TGF-ß in angiogenesis in in vitro assays using cultured endothelial cells and ex vivo metatarsal explants.

Partitioning of TSE infectivity during ethanol fractionation of human plasma.

The practice of validating processes for their capacity to inactivate a range of non-enveloped and enveloped viruses also provides confidence that plasma products will be safe from emerging viral pathogens with known aetiology. Of greater concern are diseases of unknown or poorly defined aetiology such as the group of neurological diseases collectively called the transmissible spongiform encephalopathies (TSEs), or prion diseases, for which the best known human disease is Creutzfeldt-Jakob Disease (CJD) and its variant form (vCJD). The goal of the current study was to investigate the potential for manufacturing steps used in the production of albumin and immunoglobulin products by Kistler-Nitschmann fractionation, and the utility of nanofiltration of immunoglobulin to remove TSE agents. Two different scrapie model systems were used. In the first system infectious material used for spiking was scrapie sheep brain homogenate with infectivity titres being measured in hamsters. In the second system purified scrapie agent was used (PrP fibrils) with Western blot analysis measuring reduction in the proteinase K resistant form being used as a measure of removal. The data demonstrated substantial removal of the infectious agent by the manufacturing process in both model systems although some differences were observed in partitioning of the two different infectious materials. The hamster infectivity studies were shown to be approximately 1000 fold more sensitive than the Western Blot assay. The data from both studies provide added confidence that these plasma products are safe with respect to their potential to transmit TSE.

Partial purification and properties of human brain aldehyde dehydrogenases.

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.

Aldehyde oxidizing capacity of erythrocytes in normal and alcoholic individuals.

The acetaldehyde metabolizing capacity in blood of alcoholics and nonalcoholics was investigated by an improved head-space gas chromatographic method. Great individual and interindividual variability was observed. The mean acetaldehyde oxidizing capacity of 3.51 nmoles/min/ml erythrocyte suspension in alcoholics was significantly lower than the mean of 5.20 nmoles/min/ml in nonalcoholics. Furthermore, treatment of alcoholics with aldehyde dehydrogenase inhibitors reduced the acetaldehyde oxidizing capacity significantly (mean of 1.67 nmoles/min/ml). No acetaldehyde could be detected in blood of nonalcoholics who ingested 0.25 g ethanol/kg body weight whereas levels of 2-14 microM were detected in blood of alcoholics. After disulfiram, an elevation to 7-103 microM in blood of alcoholics was observed.

The polymorphisms of alcohol and aldehyde dehydrogenase and their significance for acetaldehyde toxicity.

Evidence is growing that acetaldehyde is responsible for some toxic effects after ethanol intake. Large individual and racial differences in blood and breath acetaldehyde concentrations are observed after alcohol consumption. In many Orientals but few Caucasians extremely high blood acetaldehyde levels occur leading to an acute aldehyde syndrome also observed after treatment with aldehyde dehydrogenase inhibitors. Individuals suffering from the aversive symptoms of that syndrome will be protected from excessive drinking and the related problems. In chronic aldehydism slightly elevated aldehyde concentrations are observed possibly leading to organic injury due to the cytotoxic action of acetaldehyde. Sites exhibiting high alcohol dehydrogenase activity may specifically be affected in alcoholics.