Cyclic nucleotides and somatomedin action in cartilage.

The role of cyclic nucleotides was evaluated in the stimulation of cartilage metabolism by somatomedin-C (Sm-C). The effects of cAMP and cyclic guanosine monophosphate (cGMP) analogs on matrix synthesis were evaluated. The effects of Sm-C on tissue concentrations of these cyclic nucleotides were investigated. Likewise, the direct effects of Sm-C on the activities of cartilage adenylate cyclase, guanylate cyclase, and phosphodiesterase were determined. We found that tissue concentrations of cAMP in cartilage declined rapidly during organ culture, despite the presence of serum or Sm-C, cGMP concentrations in cartilage declined rapidly during control incubations but were augmented significantly at 30 and 60 min of incubation with the addition of serum or Sm-C. Thereafter, cGMP concentrations declined toward the levels of incubated control cartilages. Sm-C had no effect on phosphodiesterase activity. N6-Monobutyryl cAMP stimulated sulfate uptake, but dibutyryl cGMP did not. Sm-C did not stimulate adenylate cyclase in purified plasma membranes from chondrocytes, whereas it stimulated both plasma membrane and cytosol guanylate cyclase at concentrations of Sm-C as low as 10(-12) M. These data would indicate that cAMP is not the intracellular second messenger for Sm-C in cartilage. The data for cGMP are provocative and suggest it as a candidate for a second messenger mediating a portion of Sm's stimulation of cartilage metabolism.

Insulin resistance in patients with myotonic dystrophy.

Evaluation of insulin sensitivity in 12 patients with myotonic dystrophy gave results different from those found in other insulin-resistant conditions. Nine of our subjects were insensitive to exogenous insulin, but only three had elevated fasting insulin concentrations. Eight had an excessive insulin response to a glucose challenge. Monocyte insulin receptor affinity was decreased (myotonics, 1.21 +/- 0.74 X 10(9) liters per mole; controls, 2.62 +/- 1.28 X 10(9)), and this parameter correlated best with the insulin resistance. No circulating receptor antibody or insulin binding inhibitor was found. Our studies suggest that the insulin resistance seen in patients with myotonic dystrophy is related to decreased insulin receptor affinity.

Proinsulin radioimmunoassay in the evaluation of insulinomas and familial hyperproinsulinemia.

Two new radioimmunoassays for human proinsulin (hPI) have been developed and used to study patients with islet cell tumors and familial hyperproinsulinemia. Both antisera were adsorbed against human C-peptide conjugated to Sepharose, following which cross-reactivity to insulin and C-peptide was less than 0.001%. Antiserum 18D recognized the junction between the insulin B-chain and C-peptide and provided fivefold greater sensitivity than our previously reported hPI assay. Antiserum 11E recognized a determinant which includes or is adjacent to the A-chain-C-peptide junction or which is specified by the tertiary structure. In all 20 patients studied with surgically confirmed islet cell tumors, fasting plasma proinsulinlike material (PLM) was abnormal (greater than 3 SD from the mean measured in either lean or obese subjects) in both assays. This provided better discrimination than has been reported for PLM measured by gel filtration (abnormal in 13 of 14 of the present samples) with a considerably less laborious procedure. Samples from two families in which a mutant proinsulin is present in the circulation have immunoreactivity in the two assays consistent with previous identification of the molecule as an A-chain-C-peptide-linked intermediate of proinsulin conversion. The immunoreactivity of a sample from another family in which large amounts of proinsulin circulate are consistent with an intact molecule being the predominant form. This assay will be useful for confirming the diagnosis of insulin-secreting tumor in patients suspected of recurrent fasting hypoglycemia and in physiologic studies of proinsulin secretion.

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states.

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.

Clinicopathologic correlations of soluble amyloid beta-protein precursor in cerebrospinal fluid in patients with Alzheimer disease and controls.

The authors compared concentrations of soluble beta-amyloid protein precursor (s beta PP) in cerebrospinal fluid (CSF) in 45 patients diagnosed with probable Alzheimer disease (AD) and 26 normal older control volunteers. Soluble beta-amyloid protein precursor concentrations were measured in 125 CSF samples using an enzyme-linked immunosorbent assay. All subjects had Mini-Mental State Examination (MMSE) and Clinical Dementia Rating Scale (CDRS) scores and assessment of disease duration. The s beta PP concentrations in CSF in the probable AD group (mean +/- SD = 493 +/- 268 micrograms/L) were decreased significantly compared with the age-matched control group (mean = 831 +/- 302 micrograms/L; p < 0.0001). In the probable AD group, MMSE scores correlated positively with s beta PP concentrations (correlation coefficient r = 0.53, p < 0.0001), and CDRS ratings and disease duration correlated inversely with s beta PP concentrations (r = -0.59, p < 0.0001 and r = -0.479, p = 0.0006, respectively). Although the decrease in CSF s beta PP from levels found in healthy elderly controls was significant in AD subjects, there was substantial overlap. In AD, CSF s beta PP was most reduced in patients in later stages of the disease. The s beta PP concentrations reflect disease severity, but utility in differential diagnosis has not been determined.