Attenuation of muscle atrophy by an N-terminal peptide of the receptor for proteolysis-inducing factor (PIF)

Background: Atrophy of skeletal muscle in cancer cachexia has been attributed to a tumour-produced highly glycosylated peptide called proteolysis-inducing factor (PIF). The action of PIF is mediated through a high-affinity membrane receptor in muscle. This study investigates the ability of peptides derived from the 20 N-terminal amino acids of the receptor to neutralise PIF action both in vitro and in vivo. Methods: Proteolysis-inducing factor was purified from the MAC16 tumour using an initial pronase digestion, followed by binding on DEAE cellulose, and the pronase was inactivated by heating to 80°C, before purification of the PIF using affinity chromatography. In vitro studies were carried out using C2C12 murine myotubes, while in vivo studies employed mice bearing the cachexia-inducing MAC16 tumour. Results: The process resulted in almost a 23?000-fold purification of PIF, but with a recovery of only 0.004%. Both the D- and L-forms of the 20mer peptide attenuated PIF-induced protein degradation in vitro through the ubiquitin-proteosome proteolytic pathway and increased expression of myosin. In vivo studies showed that neither the D- nor the L-peptides significantly attenuated weight loss, although the D-peptide did show a tendency to increase lean body mass. Conclusion: These results suggest that the peptides may be too hydrophilic to be used as therapeutic agents, but confirm the importance of the receptor in the action of the PIF on muscle protein degradation

Cutaneous nociception and neurogenic inflammation evoked by PACAP38 and VIP

Pituitary adenylate cyclase-activating peptide-38 (PACAP38) and vasoactive intestinal peptide (VIP) belong to the same secretin–glucagon superfamily and are present in nerve fibers in dura and skin. Using a model of acute cutaneous pain we explored differences in pain perception and vasomotor responses between PACAP38 and VIP in 16 healthy volunteers in a double-blind, placebo-controlled, crossover study. All participants received intradermal injections of 200 pmol PACAP38, 200 pmol VIP and placebo into the volar forearm. Measurements included pain intensity on a visual analog scale (VAS), blood flow by laser Doppler flowmetry, visual flare and wheal. Pain intensities after PACAP38 and VIP were mild and limited to a short time of about 100 s after injection. The area under the VAS-time curve was larger following PACAP38 (P = 0.004) and VIP (P = 0.01) compared to placebo. We found no statistical difference in pain perception between PACAP38 and VIP. Skin blood flow increase, flare and wheal were larger after both PACAP38 (P = 0.011) and VIP (P = 0.001) compared to placebo. VIP induced a considerably larger increase in skin blood flow, flare and wheal than PACAP38 (P = 0.002). In conclusion, we found that peripheral nociceptive cutaneous responses elicited by PACAP38 and VIP are similar in healthy volunteers. This suggests that acute pain and vasomotor responses following intradermal injections of PACAP38 and VIP are primarily mediated by VPAC receptors

Arabidopsis thaliana encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation

The last steps of the Leu biosynthetic pathway and the Met chain elongation cycle for glucosinolate formation share identical reaction types suggesting a close evolutionary relationship of these pathways. Both pathways involve the condensation of acetyl-CoA and a 2-oxo acid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylation of the 3-malate derivative to give an elongated 2-oxo acid, and transamination to generate the corresponding amino acid. We have now analyzed the genes encoding the isomerization reaction, the second step of this sequence, in Arabidopsis thaliana. One gene encodes the large subunit and three encode small subunits of this enzyme, referred to as isopropylmalate isomerase (IPMI) with respect to the Leu pathway. Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. In contrast, the small subunits appear to be specialized to either Leu biosynthesis or Met chain elongation. Green fluorescent protein tagging experiments confirms the import of one of the IPMI small subunits into the chloroplast, the localization of the Met chain elongation pathway in these organelles. These results suggest the presence of different heterodimeric IPMIs in Arabidopsis chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism determined by the nature of the different small subunit

Hallucinogen-like actions of 2,5-dimethoxy-4-( n )-propylthiophenethylamine (2C-T-7) in mice and rats

Few studies have examined the effects of 2,5-dimethoxy-4-( n )-propylthiophenethylamine (2C-T-7) in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46369/1/213_2005_Article_9.pd