[Isolation of H. pylori proteins and support for their suitability in serologic diagnoses]. / Isolierung von H.pylori-Proteinen und Beurteilung ihrer Eignung für den Einsatz in serologischen Diagnosesystemen.

H.pylori-proteins were separated using gel chromatographic methods. These antigens were tested for their suitability to detect H.pylori-specific antibodies. A complex of two proteins (62 kDa and 30 kDa) was a strong and specific antigen. A third protein (13 kDa) was a good but nonspecific antigen. Concerning these facts we compared two often used antigen preparations for serodiagnosing H.pylori-specific antibodies (acid-glycine preparation and sarcosyl-insoluble outer membrane proteins). The sarcosyl-insoluble material contains more specific antigens and lower levels of nonspecific proteins compared to the acid-glycine preparation. Based on these results we conclude that the outer membrane preparation seems to be more suitable for the serodiagnosing of H.pylori-specific antibodies.

Multilayered keratinocyte culture used for in vitro toxicology.

Human keratinocytes are the most appropriate target cells for evaluating mechanisms of skin cytotoxicity and pharmacology of chemical agents. After having formed a confluent stratified epithelium with proliferating basal cells and differentiated cell layers, human keratinocytes were harvested after enzymatic detachment as stable, three-dimensional cell aggregates or used as adherent multilayers in microtiter plates to study the local cytotoxicity of different toxic compounds. The advantage of this test is that it uses the adequate target cells and that it evaluates both the ability of the test chemical to penetrate several cellular layers as well as the ability to interfere with cellular function. The end points are cell viability and cell metabolism, which are determined by neutral red uptake and MTT reduction, respectively. For the 13 chemicals evaluated in this study we found good correlation (r = .819) between the potency rankings of keratinocyte NR 50 values and in vivo irritancy data. There was also good agreement (r = .945) between ranking of these chemicals according to midpoint toxicity of both the 3T3 assay and the keratinocyte assay. This test system might be at the present stage a supplementation of the current test battery, which shall replace in vivo irritation tests like the Draize test.

Catalytic unit of adenlyate cyclase: purification and identification by affinity crosslinking.

The guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocardial membranes was purified approximately equal to 60,000-fold to a specific activity of 15 mumol X mg-1 X min-1 by Lubrol PX extraction, affinity chromatography, and gel permeation HPLC. The major purification (greater than 2000-fold) was achieved by affinity chromatography on forskolin-Sepharose, a method previously developed in this laboratory. The final product appeared as two major peptides of Mr 150,000 and 42,000 and one minor peptide of Mr 45,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. It is suggested that the Mr 42,000 and 150,000 components represent the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein (GS) and the catalytic unit, respectively, because upon crosslinking of a reconstituted adenylate cyclase containing the [32P]ADP-ribosylated alpha-subunit of GS (Mr, 42,000), a single radiolabeled product of Mr 190,000 appeared on NaDodSO4/polyacrylamide gels. Further identification is based on the correlation of the Mr 150,000/42,000 bands with enzymatic activity when the purified enzyme was analyzed by various chromatographic procedures.