Binding of insulin by monkey and pig hypothalamus.

Membrane preparations from monkey and pig hypothalami bound [125I]insulin specifically. The binding appeared to be greater by preparations from anterior than posterior portions of the pig hypothalamus. Binding was time dependent, and its dissociation was first order with a half-time at 22 degrees C of 14 min. Desalanine insulin was as effective as native insulin in inhibiting the binding of [125I]insulin, while proinsulin was less effective and desoctapeptide insulin still less effective in accord with their biologic activities. Binding by membranes from cortex and thalamus appeared to be less than from hypothalamus. [125I]insulin was infused into an arterial split monkey brain preparation to determine if insulin that was blood borne bound specifically to the primate hypothalamus. Half the brain was perfused with [125I]insulin alone and the other half with [125I]insulin plus an excess of unlabeled insulin. Radioautography showed specific binding of insulin localized to the median eminence, infundibular nucleus, and microvessels. Thus, the monkey and pig hypothalami bind insulin with characteristics similar to those reported for known target tissues for insulin. Furthermore, insulin from the blood stream binds to specific anatomical structures in the hypothalamus of the monkey.

Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Effects of cognitive coping skills training on coping strategies and experimental pain sensitivity in African American adults with sickle cell disease.

The present study examined whether training in cognitive coping skills would enhance pain coping strategies and alter pain perception in adults with sickle cell disease (SCD). Sixty-four African Americans with SCD were randomly assigned to either a cognitive coping skills condition (three 45-min sessions in which patients were trained to use 6 cognitive coping strategies) or a disease-education control condition (three 45-min didactic-discussion sessions about SCD). Pain sensitivity to calibrated noxious stimulation was measured at pre- and posttesting, as were cognitive coping strategies, clinical pain, and health behaviors. Results indicated that, compared with the randomly assigned control condition, brief training in cognitive coping skills resulted in increased coping attempts, decreased negative thinking, and lower tendency to report pain during laboratory-induced noxious stimulation.

Quantitative analysis of metabolism of hepatic triglyceride in ethanol-treated rats.

An acute intraperitoneal injection of ethanol (0.7 or 2.1g/kg body wt.) causes the reversible, dose-dependent accumulation of hepatic triglyceride in rats. By using a pulse of [14C]palmitate injected into a tail vein, it was found that ethanol (2.1g/kg)had no effect on the flux of unesterified fatty acid of serum (4.3mumol/min per 100g body wt.). However, either dose increased the fraction of the total flux going to liver from 0.16 to0.27 as rapidly as could be measured (30s), and it remained elevated until all ethanol had been cleared from the blood. The fraction of the total radioactivity in lipids of liver that was in triglyceride increased linearly for 1 h from 30 to 50% and there was a simultaneous decrease in phospholipid from 60 to 40%. The rate of synthesis of hepatic triglyceride derived directly from unesterified fatty acid of serum was calculated by using the flux rate of unesterified fatty acid in serum, the fractional hepatic uptake of this flux, and the percentage of liver fatty acid esterified to triglyceride. This contribution is related to the total synthetic rate of hepatic triglyceride (rate of accumulation+rate of release) to determine quantitatively how much of the developing fatty liver is attributable to increased uptake of unesterfied fatty acid of serum. At the higher dose of ethanol, about half of the accumulating triglyceride is derived from this source, whereas with the lower dose of ethanol it can account for all of the build-up.

Mechanism of increased hepatic uptake of unesterified fatty acid from serum of ethanol-treated rats.

Studies were made on the mechanism by which livers of ethanol-treated rats take up an increased fraction of the total flux of unesterified fatty acid in serum. It was found that ethanol (0.7g/kg) causes a twofold rise in the serum content of liver, and that this serum is in rapid equilibrium with the general circulation. The fractional hepatic uptake from serum of group of compounds with varying uptake mechanisms and metabolic fates was studied in control and ethanol-treated animals. All the compounds tested, including unesterified fatty acid, showed an enhanced uptake when ethanol was given. For one of the compounds, carbon tetrachloride, a dose/response relationship was established between the amount administered, the amount taken up by liver, and the amount metabolized. These findings were interpreted to mean that this dose of ethanol causes the liver to receive an increased flow of blood, and as a result all compounds present and capable of being taken by liver are taken up at an increased rate. Hepatic blood flow was measured by a technique that monitors the rate of clearance of a colloidal lipid emulsion. It was found that ethanol increased hepatic blood flow by about 60%. This effect of ethanol on hepatic blood flow provides an explanation for the fatty liver and the synergistic effect between an acute dose of ethanol and carbon tetrachloride. A hypothesis to explain why a moderate dose of ethanol causes triglyceride to accumulate in liver is presented.