Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10^-6 and 10^-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ~10^-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids.     

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle.

Ca2+-activated Z-disk-removing activity in the P0-40 crude muscle extracts described by Busch et al. (Busch, W. A., Stromer, M. H., Goll, D. E., and Suzuki, A. (1972), J. Cell Biol. 52, 367) was purified from porcine skeletal muscle extracts by using five column chromatographic procedures in succession: (1) 6% agarose; (2) DEAE-cellulose; (3) Sephadex G-200; (4) DEAE-cellulose with a very shallow gradient; (5) Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca2+-activated proteolytic activity, so Z-disk-removing activity in the P0-40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The five column chromatographic procedures produced a 140-fold increase in specific activity of the Ca2+-activated proteolytic enzymic activity; because preparation of the P0-40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure described here produces a 17 800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 mug of CAF per g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25-0.76 mug of purified CAF per g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HC1 buffer but migrated as two bands with molecular weights of 80 000 and 30 000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80 000- and 30 000-dalton subunits make up 85 to 90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.     

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.     

Evaluation of burn blister fluid

Although edema is evident immediately after a burn, the diffusion of nutrient chemical constituents of the body is not impaired. Blister fluid, not unlike plasma or serum, contained all substances found in the body, including parenterally administered penicillin. The elevation of potassium and the cation to anion imbalance is primarily due to the Na/K cellular pump malfunction, and the destruction of the permeability of the cell membrane is most likely a direct result of complement and other cellular enzymes, which include the prostaglandins and thromboxanes. The elevated SGOT, CPK, and LDH indicated severe trauma to the cells in the immediate area of burn and possibly to the skeletal muscle. The presence of immunoglobulins indicated that high-molecular-weight proteins diffuse equally well during this edematous phase (IgM, 900,000; IgG, 190,000). Evidence of this nature strongly suggests that the integrity of the burn blister by maintained.