Phosphorylation and Palmitoylation of the Human D2L Dopamine Receptor in Sf9 Cells

Abstract: We have expressed and biochemically characterized the human D2_long (D2_L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2_L receptors expressed in mammalian cell lines. Dopamine binding to D2_L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2_L receptor-specific antibody identified two major protein species at ∼44 kDa and at ∼93 kDa in immunoblots, suggesting the presence of D2_L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2_L receptor prelabeled with ³²P_i or [³H]-palmitate. These results constitute the first direct evidence for D2_L receptor phosphorylation and palmitoylation.     

Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.     

Strength, skeletal muscle composition, and enzyme activity in multiple sclerosis

Kent-Braun, J. A., A. V. Ng, M. Castro, M. W. Weiner, D. Gelinas, G. A. Dudley, and R. G. Miller. Strength, skeletal musclecomposition and enzyme activity in multiple sclerosis. J. Appl. Physiol. 83(6):1998-2004, 1997.This study examined functional, biochemical, andmorphological characteristics of skeletal muscle in nine multiplesclerosis (MS) patients and eight healthy controls in an effort toascertain whether intramuscular adaptations could account for excessivefatigue in this disease. Analyses of biopsies of the tibialis anteriormuscle showed that there were fewer type I fibers (66 ± 6 vs. 76 ± 6%), and that fibers of all types were smaller (average26%) and had lower succinic dehydrogenase (SDH; average40%) and SDH/-glycerol-phosphate dehydrogenase (GPDH) butnot GPDH activities in MS vs. control subjects, suggesting that musclein this disease is smaller and relies more on anaerobic thanaerobic-oxidative energy supply than does muscle of healthyindividuals. Maximal voluntary isometric force fordorsiflexion was associated with both average fiber cross-sectionalarea (r = 0.71, P = 0.005) and muscle fat-free cross-sectional area by magnetic resonance imaging(r = 0.80, P < 0.001). Physical activity,assessed by accelerometer, was associated with average fiber SDH/GPDH(r = 0.78, P = 0.008). There was a tendency forsymptomatic fatigue to be inversely associated with average fiber SDHactivity (r = 0.57,P = 0.068). The results of thisstudy suggest that the inherent characteristics of skeletal musclefibers per se and of skeletal muscle as a whole are altered in thedirection of disuse in MS. They also suggest that changes in skeletalmuscle in MS may significantly affect function.

     

SIALYLTRANSFERASES IN RAT BRAIN: INTRACELLULAR LOCALIZATION AND SOME MEMBRANE PROPERTIES

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes.
The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non-ionic detergent mixture, Triton CF-54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM_1a, 700; exogenous DS-fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza-tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.     

Cellular and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum.

Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex.