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.     

The α2(XI) collagen gene lies within 8 kb of Pb in the proximal portion of the murine major histocompatibility complex

A number of serious hereditary disorders are now known to be associated with defective expression of collagen genes, and these findings have underscored the important and varied roles that the collagen family of genes must play during normal mammalian development. Although the activities of genes encoding the quantitatively major types of collagen are fairly well characterized, functions of the many minor types of collagen remain a matter of speculation. As a first step toward a functional analysis of type XI collagen, a member of this class of poorly understand minor collagen proteins which is expressed primarily in hyaline cartilage, we have used human probes for the gene encoding the protein's 2-subunit (COL11A2) to isolate and map homologous murine DNA sequences. Our results demonstrate that Col11a-2 is embedded within the major histocompatibility complex (MHC), within 8.4 kb of the class II pseudogene locus, Pb, and confirm that human and murine 2(XI) collagen genes are located in very similar genomic environments. The conserved location of these genes raises the possibility that type XI collagen genes may contribute to one or more of the diverse hereditary disorders known to be linked to the MHC in mouse and human.     

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.

     

Exfoliative cytology in the evaluation of interferon treatment of cervical intraepithelial neoplasia

Local interferon injection in four patients with cervical intraepithelial neoplasia (CIN) regularly elicited progressive regression of the lesions. The response was observed with exfoliative cytology after each injection, guided by colposcopic examination. The cytologic changes showed a cytocidal effect mainly on the dyskaryotic cells, preceded by cellular degeneration not unlike that of nonspecific inflammation and accompanied by an increase in neutrophil infiltration. The cytologic response was closely correlated with partial or complete clinical regression based on the absence of viable or degenerated dyskaryotic cells in the cervical smears. Three patients showed complete clinical regression after treatment. One patient showed recurrent viable dyskaryotic cells when the dosage was reduced, and treatment was suspended temporarily although her lesion had regressed completely after five injections. Clinical recurrence was noted one week after viable dyskaryotic cells reappeared in her smears. These observations suggest that cytology may be a useful means of monitoring interferon treatment in CIN.