Impaired cAMP-mediated gene expression and decreased cAMP response element binding protein in senescent cells.

The capacity of various growth factors to induce c-fos expression is diminished with senescence. Because adenosine 3',5'-cyclic monophosphate (cAMP)-mediated responses are also blunted with aging, we wondered whether cAMP-induced c-fos gene expression might be impaired with senescence. Using IMR fibroblasts, we found that prostaglandin E1 (PGE1) and forskolin, stimulators of cAMP accumulation in young and senescent cells, increased abundance of c-fos and junB mRNA more in young than senescent cells. The abundance of the cAMP response element binding protein (CREB), a transcription factor which enhances gene expression when phosphorylated by protein kinase A, was markedly decreased in both whole cell and nuclear extracts of senescent cells, in both Western blotting and in gel retardation assays. Also, PGE1-induced phosphorylation of CREB by protein kinase A was markedly attenuated in senescent cells. There is a marked decrement in expression of CREB with senescence, and the results suggest the possibility that the diminished expression of CREB may contribute to altered cAMP-mediated regulation of gene expression with senescence.

Phosphorylation of cyclic AMP response element-binding protein and induction of c-fos gene expression on withdrawal from chronic treatment with carbachol in NG108-15 cells.

Alterations in adenylyl cyclase activity in cultured cells after prolonged exposure to drugs such as morphine have been extensively studied as models for drug tolerance and withdrawal. NG108-15 cells develop increased intracellular cAMP concentrations after abrupt withdrawal from chronic treatment with the muscarinic cholinergic agonist carbachol. To determine whether this withdrawal-induced increase in cAMP modifies gene expression, we studied phosphorylation of the cAMP response element-binding protein (CREB) and expression of the c-fos gene, known to contain a cAMP response element, in NG108-15 cells after abrupt withdrawal from chronic treatment with carbachol. Prostaglandin E1, which activates adenylyl cyclase, caused concentration-dependent increases in the phosphorylation of CREB and in the abundance of c-fos mRNA. These changes occurred with small increments in cAMP accumulation. In cells treated with carbachol for 48 hr, induction of withdrawal with the muscarinic antagonist atropine led to a small increase in intracellular cAMP concentration but an 11.6-fold increase in the phosphorylation of CREB and a 3.4-fold increase in accumulation of c-fos mRNA. The adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, which attenuated the chronic carbachol-induced increase in cAMP concentration, prevented the increased phosphorylation of CREB and the enhanced accumulation of c-fos mRNA during atropine-induced withdrawal. These results indicate that expression of the c-fos gene is induced by the small increments in cAMP concentration that can occur in cells on withdrawal from chronic treatment with drugs such as muscarinic agonists.

Chymotrypsin-reactive antibodies in insulin-dependent diabetes mellitus.

In a previous study (Frazier et al., 1990), it was demonstrated that two patients with type 1 (insulin-dependent) diabetes mellitus had antibodies in their serum which reacted with four 29 kDa pancreas-specific proteins on two-dimensional immunoblots. This paper reports on the purification and identification of these pancreatic proteins. The protein with the pI closest to pH7 was purified through the use of ammonium sulfate fractionation and ion-exchange chromatography. Gel filtration chromatography established that the protein's molecular weight was closer to 25 kDa. Amino acid composition and sequence analyses demonstrated homology between the protein and chymotrypsin. It is suggested that an abnormal regulation of chymotrypsin activity might be related to antibodies formed in some diabetic patients.

Serum autoantibodies in type 1 (insulin-dependent) diabetes mellitus: identification of reactivity against a 29 kd pancreas-specific protein.

Serum samples from patients with type 1 (insulin-dependent) diabetes mellitus and controls were incubated with two-dimensional Western immunoblots of pancreas and other tissues. Two out of 26 (8%) of the diabetics and 0 out of 45 of the controls demonstrated reactivity against four pancreas-specific proteins with identical molecular weights of 29,000 daltons and different isoelectric points ranging from pH 7.0-8.0. It is concluded that 29 Kd autoantibody reactivity is not a major marker for type 1 diabetes, but may help identify a subgroup of type 1 diabetics.

Human antiidiotypic antibody against opiate receptors.

Sera containing antibodies to beta-endorphin from 2 patients with major depressive disorder were shown to have antidiotypic antibodies that specifically inhibited reactivity between anti-beta-endorphin IgG and beta-endorphin. Autologous and homologous antiidiotypic anti-anti-beta-endorphin IgG antibodies were isolated by affinity chromatography. The purified antiidiotypic antibody did not bind beta-endorphin but competed with [125I]beta-endorphin for rat brain opiate receptors. Normal IgG that was similarly treated had negligible competitive effects. The antibody bound to the membrane preparation; such binding was inhibited by opiate receptor ligands. Binding of the antiidiotype to a 60,000-dalton protein from rat brain was detected by Western immunoblot analysis. This protein corresponds in molecular weight to proteins proposed to be components of opiate receptors. These findings imply that immune reactivity to neuropeptides could contribute to psychiatric impairment.

Geometric modeling of the carotid bifurcation in humans: implications in ultrasonic Doppler and radiologic investigations.

A geometric representation of the carotid bifurcation is presented with data obtained from biplane angiograms of normal branches and branches exhibiting less than 5% vessel diameter reduction. Three features are identified that are of importance in the interpretation of ultrasonic Doppler velocity information and in the design of engineering flow models for evaluation of carotid branch hemodynamics: the variability of the bifurcation angles, the degree of tortuosity, and the nonplanar nature of the branches. In addition, data collected demonstrate the potential usefulness of the common carotid artery as a reference diameter in evaluating stenosis of the carotid bulb.