The murine agouti gene encodes for a novel 131 amino acid protein. The sequence includes a 22 residue putative secretion signal, an internal basic region, and a C-terminal domain containing 10 cysteines. Agouti has been found to antagonize the binding of certain pro-opiomelanocortin peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), to the murine melanocortin-1 receptor (MC1-R). We report the purification of a secreted murine agouti to homogeneity by a two-step procedure from baculovirus-infected Trichoplusia ni (T. ni). The protein is glycosylated and exhibits competitive, high-affinity antagonism (Ki = 0.8 nM) versus alpha-MSH in cell-based assays employing B16F10 cells. Association state analysis by analytical ultracentrifugation reveals that agouti exists in a monomer--dimer plus aggregate equilibrium at low micromolar concentrations. Data from secondary structure studies indicate that the protein is highly stable to thermal denaturation. Enzymatic digestion to probe disulfide bond arrangement yielded a discrete C-terminal (Val 83-Cys 131) domain. The isolated highly cysteine-rich C-terminal domain retains alpha-MSH antagonism equipotent with mature agouti. This bioactive domain contains all 10 cysteines which exhibit sequence homology when aligned with several conotoxins.

OBJECTIVE: To test the hypothesis that a melanocortin agonist can reverse obesity and insulin resistance in mice overexpressing the agouti protein. EXPERIMENTAL MODEL: Mice overexpressing the agouti protein either by transgene introduction (b b-actin promotor) or by mutation (A y ). DESIGN: NDPMSH was tested for pharmacokinetic suitability. NDPMSH at various doses was administered subcutaneously twice a day for 2 ± 3 weeks. MEASUREMENTS: Fur pigmentation, various fatness parameters (core temperature, fat pad weight and body weight), blood glucose and hormones, fatty acid synthase measurement. RESULTS: NDPMSH caused fur pigmentation and core temperature changes, but failed to affect any metabolic parameters in agouti-dependent manner. CONCLUSION: NDPMSH, as a representation melanocortin agonist, does not compete with agouti in reversing agoutidependent metabolic effects. This suggests that 1) agouti works via a receptor other than a melanocortin receptor to mediate its metabolic effects, 2) agouti-dependent metabolic effects are mediated through melanocortin receptors but not via antagonism of these receptors, or 3) NDPMSH is pharmacodynamically an inappropriate molecule for these types of studies.

Previous clinical studies of blunt trauma patients with severe brain injuries have demonstrated that emergency department vital signs failed to consistently identify life-threatening abdominal injury. One hypothesis to explain this is that bradycardia and systemic hypertension from brainstem injury (the Cushing response) may mask the tachycardia and hypotension ordinarily manifested by hemorrhagic hypovolemia. This would result in inappropriately normal or near-normal emergency department vital signs for otherwise clinically apparent hypovolemia. To test this hypothesis, splenectomized dogs (n = 9) were phlebotomized to a systolic blood pressure (SBP) of 60 mm Hg. Subsequently, intracranial pressure (ICP) was artificially elevated in a controlled, incremental fashion. From a mean SBP of 58.4 +/- 3.9 mm Hg at a baseline ICP of 8.1 +/- 4.2 mm Hg, increases in ICP of only 20 mm Hg significantly raised SBP (in some animals). When ICP reached 70 mm Hg, mean SBP reached 95.1 +/- 8.7 mm Hg (p < 0.001) in spite of profound hemorrhagic hypovolemia. In all subjects, the tachycardia that accompanied hypovolemia tended towards normal with incremental increases in ICP. However, this did not reach statistical significance. In response to elevations in ICP, this hypovolemic canine model displayed normalization of SBP with variable changes in heart rate. These changes could mask hemorrhagic hypotension in humans sustaining multiple system trauma. These experimental data support clinical studies advocating immediate definitive abdominal evaluation in unconscious blunt trauma patients, regardless of vital signs.

Most trauma directors consider MR important in the acute evaluation of spinal trauma and, to a lesser extent, for traumatic brain injury. Despite these opinions, the vast majority of these centers reported only "rare" to "occasional" use of MR in the setting of acute CNS trauma. Our results show that most TCs have on-site and continuously available MR facilities capable of cardiac and pulmonary monitoring. Other factors such as the higher relative cost of MR may be responsible for the discrepancy between the perceived value and the actual utilization of MR imaging in the setting of CNS trauma.