A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. A biochemical analysis and their ability to inhibit the enzyme complex.

A library of monoclonal antibodies to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC 1.2.4.1; E1) subunit is reported. 21 monoclonal antibodies were generated, and 20 were investigated, of which 9 were elicited to PDHc and 11 to pure E1 subunit; 19 were of the IgG1 isotype and one of the IgG3 isotype. According to an enzyme immunoassay, all 20 of the monoclonal antibodies bound the PDHc, and 17 bound the E1 subunit. According to Western blot analysis, 14 of the 19 monoclonal antibodies bound to the E1 subunit. The monoclonal antibodies inhibited PDHc from 0 to > 98%. The six monoclonal antibodies that displayed greater than 30% inhibition of E. coli PDHc were unable to inhibit porcine heart PDHc nor did they bind porcine heart PDHc according to dot blot analysis. Radiolabeling gave binding constants ranging from 5 to 10 x 10(8) M-1 on these six monoclonal antibodies, with greater than 80% of maximal inhibition achieved in less than 1 min. One of the six, 18A9, gave > 98% inhibition, required two antibodies/E1 subunit for maximum inhibition, and was shown to be a non-competitive inhibitor. Monoclonal antibody 15A9 was shown to counteract GTP-induced inhibition, while 1F2 influenced the conformation of E1, allowing two antibodies, which did not previously bind E1, to bind to it. A new mechanism-based kinetic assay is presented that is specific for the E1 component of 2-keto acid dehydrogenases. This assay confirmed that the three most strongly inhibitory monoclonal antibodies specifically inhibited the E1 function while antibody 1F2 led to enhanced activity, suggesting an induced conformational change in PDHc or in E1.

Effects of spaceflight on rat pituitary cell function.

The secretory capacity of growth hormone (GH) and prolactin (PRL) cells prepared from rats flown in space on the 12.5-day mission of COSMOS 1887 and the 14-day mission of COSMOS 2044 was evaluated in several postflight tests on Earth. The results showed statistically significant and repeatable decrements in hormone release, especially when biologic (rather than immunologic) assays were used in the tests. Significant and repeatable intracellular changes in GH cells from the flight animals were also found; most important were increases in the GH-specific cytoplasmic staining intensities and cytoplasmic areas occupied by hormone. Tail suspension of rats for 14 days, an established model for mimicking musculoskeletal changes in rats flown in space, resulted in some changes in GH and PRL cell function that were similar to those from animals flown in space. Our results add to a growing body of data that describe deconditioning of physiological systems in spaceflight and provide insights into the time frame that might be required for readaptation of the GH/PRL cell system on return to Earth.

Heterogeneity in mammotrophs prepared from diethylstilbestrol-induced prolactinomas.

Pituitary glands from ovariectomized F-344 rats bearing diethylstilbestrol-containing capsules for 70-100 days were dissociated and separated into three size classes by unit gravity sedimentation. The average percentage of large mammotrophs in fraction I was 86 +/- (SE) 2%, the percentage of intermediate-sized mammotrophs in fraction II was 86 +/- 4%, and the percentage of small mammotrophs in fraction III was 72 +/- 3%. By electron microscopy, cells in fractions I and II contained cytoplasmic secretory granules, whereas those in fraction III were either sparsely granulated or agranular. Cells retained their morphology after culture. Biological (B) and immunological (I) assays of PRL released into the medium after 24-h culture both showed that fraction I and II cells released more hormone than fraction III cells. The B/I ratios of released PRL were consistently greater than 1. Analysis of the separated mammotrophs by flow cytometry indicated major differences in light scatter profiles, both before and after culture. Since these differences have been shown to reflect secretory granule content, changes in perpendicular light scatter after culture were interpreted to indicate that small mammotrophs accumulated granules, whereas some of the large mammotrophs lost granules. Results from a cell blot assay indicated that fraction I and II cells secreted more hormone than fraction III cells. This same rank order was found when live cells were stained and analyzed for surface PRL. However, the reverse plaque assay yielded different information, viz. more plaques were found around fraction III cells than in fraction I or II cells. Finally, PRL contained in alkaline extracts of fraction I, II, and III cells was analyzed by Western blotting after electrophoresis under nondenaturing, denaturing, or denaturing and reducing conditions. Fraction III cells contained a prominent and unique PRL variant that had low mobility in native gels and an apparent mol wt in the range of 10-14K. After 24 h the culture medium from fraction III cells also contained this low mol wt variant. Our data indicate considerable heterogeneity in form and function within the mammotroph population of diethylstilbestrol-induced prolactinomas.

Cell bioprocessing in space: applications of analytical cytology.

Cell bioprocessing in space consists of the preparation, cultivation, purification and investigation of cells and their products in the microgravity environment of orbital space flight. Inertial acceleration is used as an independent variable to explore the limits of specific bioprocessing functions, such as cell growth and secretion, gravity-dependent phenomena in cell bioreactors, cell fusion, the influence of thermal convection on processes at cellular dimensions, the electrophoretic separation of cell subpopulations and subcellular particles, and two-phase partitioning of cells, bioparticles, and macromolecules. Analytical cytology techniques are under development for on-orbit application to future cell growth and separation experiments, such as those anticipated in the Space Station era.

Changes in pituitary growth hormone cells prepared from rats flown on Spacelab 3.

Anterior pituitaries from "small" (250 g) and "large" (400 g) rats flown on the 7-day Spacelab 3 mission were pooled and trypsinized into two single-cell suspensions. Compared with ground-based controls, flight cells appeared to contain more intracellular growth hormone (GH) but release less GH over a 6-day culture period. After implantation into hypophysectomized rats, both sets of flight cells released only 50% of the GH compared with the control cells. Glands from large flight rats contained 44% somatotrophs compared with 37% for controls; small animals showed no difference. There were no striking differences in somatotroph ultrastructure between cells in the four groups. Western blot analysis indicated that there were no major differences in immunoactive GH variants. High-performance liquid chromatography fractionation of culture media indicated that small flight cells released much less of a high-molecular weight variant rich in GH bioactivity. The results suggest that GH cells from rats exposed to microgravity may experience secretory dysfunction. The possibility that this occurs directly at the pituitary cell level is discussed.