Insulin prohormone processing, distribution, and relation to metabolism in Aplysia californica.

The first Aplysia californica insulin gene is characterized and its proteolytic processing from prohormone to final peptides elucidated using a combination of biochemical and mass spectrometric methods. Aplysia insulin (AI) is one of the largest insulins found, with a molecular weight of 9146 Da, and an extended A chain compared with other invertebrate and vertebrate insulins. The AI prohormone produces a series of C peptides and also a unique N-terminally acetylated D peptide. AI-producing cells are restricted to the central region of the cerebral ganglia mostly within the F and C clusters, and AI is transported to neurohemal release sites located on the upper labial and anterior tentacular nerves. The expression of AI mRNA decreases when the animal is deprived of food, and injections of AI reduce hemolymph glucose levels, suggesting that the function of insulin-regulating metabolism has been conserved.

The Aplysia mytilus inhibitory peptide-related peptides: identification, cloning, processing, distribution, and action.

Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

Evidence for eosinophil activation in cancer patients receiving recombinant interleukin-4: effects of interleukin-4 alone and following interleukin-2 administration.

Interleukin-4 (IL-4) is a T-cell-derived cytokine that may mediate murine tumor rejection through the activation of host eosinophils. In association with a Phase I clinical trial of IL-4 in cancer patients, we have examined changes in eosinophil counts and characterized systemic eosinophil degranulation. As previously reported, IL-4 administration induced a modest eosinophilia in all 17 evaluated patients. Here, we report that IL-4 therapy induced systemic eosinophil degranulation based on increases in serum major basic protein (MBP) (P = 0.018) and urine MBP (P = 0.031). The increase in serum MBP was IL-4 dose dependent (P = 0.001). Following the highest dose (600 microgram/m2/day) of IL-4 administered, mean serum MBP levels were >2000 ng/ml. Skin biopsies of rashes from patients receiving IL-4 revealed MBP deposition. Sera from eight patients receiving IL-4 at 360 and 600 microgram/m2/day exhibited eosinophil survival-enhancing activity (on days 3, 5, 7, and 9) significantly above pretreatment (on day 1) activity (P values 0. 0469, 0.0039, 0.0395, and 0.0313, respectively). This enhanced eosinophil survival could be neutralized by antibodies to IL-5, granulocyte-macrophage-colony-stimulating factor, and IL-3. The eosinophil activation demonstrated in this trial may be relevant to the clinical effects of IL-4 in cancer patients. Furthermore, an association between IL-4 and eosinophil activation should be explored in other disease states.

PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia.

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.