Steroid-responsive myopathy with deficient chondroitin sulfate C in skeletal muscle connective tissue.

A 71-year-old man developed severe limb, bulbar, and respiratory weakness over 18 months. A muscle biopsy showed only a moderate degree of type 2 atrophy, but immunocytochemistry showed absence of chondroitin sulfate C glycosaminoglycan in the endomysium. Prednisone produced a marked increase in strength. Diffuse loss of endomysial chondroitin sulfate C was a feature of this treatable myopathy with severe weakness, but few pathologic changes.

The transient nicotinic stimulation of tyrosine hydroxylase gene transcription in bovine adrenal chromaffin cells is independent of c-fos gene activation.

The nicotinic agonist dimethylphenylpiperazinium (DMPP) transiently stimulates tyrosine hydroxylase (TH) gene transcription in cultured bovine adrenal chromaffin cells (Craviso et al., J. Neurochem., 59 (1992) 2285-2296). The present studies examined the mechanism of this stimulation, exploring the hypothesis that c-fos- and/or cyclic AMP-related mechanisms are involved. As determined by nuclear run-on assay, exposure of chromaffin cells to DMPP (1 microM) induced c-fos and TH gene transcription fivefold and twofold, respectively. Nitrendipine (20 microM) blocked both responses, indicating a similar dependency of each on extracellular calcium. Both c-fos and TH gene transcription rates were also elevated by entry of calcium due to the presence of the calcium ionophore A23187 (5 microM). Comparison of the time dependence of the DMPP stimulation of c-fos and TH gene transcription revealed similar time courses. Both were rapid and transient, peaking within 10-30 min of nicotinic receptor occupancy and returning to control values by 1 h. This simultaneous activation of the TH and c-fos genes indicates that Fos induction cannot be responsible for the stimulation of TH gene transcription. This conclusion was further supported by a failure of anisomycin (100 microM) pretreatment of chromaffin cells, which blocked protein synthesis 99%, to have any effect on either the rapid stimulation of TH gene transcription or the length of time that the TH gene was activated by DMPP. Thus, neither Fos nor other high turnover-rate transcription factors appear to be responsible for the stimulation, or return to control level, of TH gene activity following nicotinic stimulation of chromaffin cells. In other experiments, treating chromaffin cells with a combination of maximally effective concentrations of DMPP and forskolin was found to produce no greater stimulation of TH gene transcription than either agent alone, suggesting that DMPP acts through the same mechanism as forskolin. Taken together, these results support the conclusion that the mechanism of TH gene activation in chromaffin cells by DMPP involves a cyclic AMP-dependent process and not the induction of transcription factors such as Fos.

Nicotinic cholinergic regulation of tyrosine hydroxylase gene expression and catecholamine synthesis in isolated bovine adrenal chromaffin cells.

Isolated bovine adrenal chromaffin cells were used to study the nicotinic regulation of tyrosine hydroxylase (TH) gene expression. Continuous exposure of the cells to carbachol or the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) produces a time- and concentration-dependent increase in TH enzyme activity, whereas muscarine has no effect. DMPP at 1 microM (EC50 = 0.3 microM) elicits a two- to threefold elevation of both TH activity and TH immunoreactive protein level after 3-5 days in the presence of 2.5 mM calcium; the increase in enzyme levels is significantly less at lower extracellular calcium levels. The rate of hydroxylation of tyrosine to dopamine (DA) in intact cells, an index of endogenous TH activity, increases in parallel with the rise in TH levels. The TH mRNA level is elevated before the increase in protein levels. As determined by nuclear run-on assays, TH gene transcription is stimulated two- to threefold within 30 min of addition of 1 microM DMPP to the cells; transcription returns to basal levels by 2 h. Nitrendipine (20 microM) blocks the stimulation of transcription by DMPP. Pretreatment of the cells with cycloheximide (5 microM) does not prevent the DMPP stimulation of transcription. Forskolin (10 microM) also increases TH transcription (fourfold in 15 min) by a mechanism that is not blocked by cycloheximide. These results show that nicotinic receptor stimulation increases TH mRNA synthesis, TH protein levels, and TH activity in a calcium-dependent manner. Furthermore, the nicotinic influence on TH gene expression does not appear to require the synthesis of a protein factor for its effects. That in situ DA synthesis rates are elevated consequent to the rise in TH levels demonstrates that TH induction serves as a mechanism for enhancing the catecholamine-synthesizing capacity of the chromaffin cell on a long-term basis.

Exposure of antigenic sites during immunization. Monoclonal antibodies to monomer of lactose repressor protein.

Monoclonal antibodies specific for the lactose repressor protein have been purified from three mouse hybridoma cell lines, and ascitic fluids from five other cell lines producing repressor antibodies have been assayed for immunoglobulin subclass and antigenic specificity. The chymotryptic core region (amino acids 57-360) of the repressor reacted with all antibodies examined, while no reaction with the NH2-terminal domain (1-56) could be detected. All of the purified antibodies and ascitic fluids reacted with the carboxyl-terminal fragment (amino acids 281-360) produced by cyanylation and base-catalyzed cleavage at the cysteine residues. Although none of the purified antibodies associated with native, tetrameric lac repressor, reaction was observed with repressor which had been denatured or dissociated into monomers by treatment with low levels of sodium dodecyl sulfate. Additionally, a mutant repressor which exists as a monomer in solution reacted with the antibodies in the absence of any denaturing treatments. These data indicate the carboxyl-terminal region is inaccessible in the intact repressor tetramer and further suggest that denaturation/dissociation of a protein during the initial immunologic challenge may result in the production of monoclonal antibodies to antigenic areas of the protein which are not exposed in the native conformation.