Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors

Fluorescence resonance energy transfer (FRET) with fluorescent proteins is a powerful method for detection of protein-protein interactions, enzyme activities and small molecules in the intracellular milieu. Aided by a new violet-excitable yellow-fluorescing variant of Aequorea victoria GFP, we developed dual FRET-based caspase-3 biosensors. Owing to their distinct excitation profiles, each FRET biosensor can be ratiometrically imaged in the presence of the other.     

Tissue distribution of avian pancreatic polypeptide-degrading activity

Degradation of avian pancreatic polypeptide (APP) by subcellular fractions from homogenates of chicken kidney, liver, and brain was characterized in this study. Chicken kidney cytosol exhibited the highest degrading activity of all kidney subcellular fractions studied including nuclear, mitochondrial, and microsomal. The cytosolic kidney APP-degrading activity was inhibited in a dose-dependent manner by bacitracin, serine protease inhibitors, and dithiothreitol, and eluted in the void volume of a Sephadex G-100 column, indicating that it is a soluble, serine protease-like activity with a Mr greater than 100,000 kDa and with some dependence on disulfide bonds. Soluble cytosol fractions from chicken liver, kidney, and brain all exhibited greater APP-degrading activity than that of corresponding membrane fractions and, furthermore, were similar in activity between one another. It is concluded that APP degradation by tissue homogenates occurs via a soluble, cytosolic protease which is inhibited by selected serine protease inhibitors; the activity does not differ among liver, kidney, and brain, three tissues which show different receptivity for APP.     

Effects of hypophysectomy on plasma levels of pancreatic polypeptide (APP) and insulin in adult chickens

Adenohypophysectomy (hypox) was carried out in adult chickens in an attempt to assess what role, if any, the anterior pituitary gland plays in maintaining basal levels of plasma insulin (IRI) and avian pancreatic polypeptide (APP) both before and immediately after a fast-refeed regimen. Each bird was tube-fed 61 gms twice daily, body weights were taken daily, and blood samples drawn daily just before the second feeding. All birds were fasted for 24 hr on days 4-5, another blood sample taken, and then refed the usual gruel. Blood samples were taken at 5,15,30,90 and 180 min after refeeding. Hypox caused an immediate and sustained decrease in plasma IRI and a significant but transient increase in plasma APP which lasted 3-4 days before returning to normal; plasma glucose was marginally decreased. Refeeding resulted in a trend of less response (increase) in all three parameters studied in the hypox group. It is suggested that in chickens, hypox may lead to a release phenomenon from a normal inhibitory role which the anterior pituitary gland plays on APP release.     

Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites

Binding isotherms were constructed for the binding of synthetic tetrapeptide and pentapeptide fragments to membranes prepared from chicken cerebellar tissue. Both the tetrapeptide (FMRFamide), which was originally isolated from ganglia of mollusks, and the pentapeptide (LPLRFamide) previously isolated from chicken brain are known to increase blood pressure and modulate brain neurons in rats. The C-terminal dipeptide sequences of the two peptides are identical and both show similarity to the dipeptide sequence established for the pancreatic polypeptide (PP) family. Specific high-affinity binding sites exist for the latter peptide, sites which are competed for (though with less affinity) by neuropeptide Y (NPY). Affinity for cerebellar membranes was virtually equivalent for the synthetic peptide LPLRFamide and FRMFamide; the binding affinities (IC50) of all fragments tested (C-terminal pentapeptides of avian PP and NPY, and FMRFamide and LPLRFamide) fell in the same approximate range. Since the N-terminal residues of FMRFamide and LPLRFamide are not homologous with equivalent residues of APP or NPY, our results indicate that only Arg-Tyr-NH2 or Arg-Phe-NH2 sequences are necessary for binding of the carboxy terminus peptides of the PP family. In this respect, these sequences are functionally equivalent.     

Pancreatic hormones, insulin/glucagon molar ratios, and somatostatin as determinants of avian carbohydrate metabolism

The endocrine pancreas of birds contains cell populations similar, if not identical, to those found in mammalian pancreata, although the topographical distributions of these cell types differ to some extent. Insulin-secreting (B) cells, glucagon-secreting (A) cells, somatostatin-secreting (D) cells, and pancreatic polypeptide-secreting (PP or F) cells are distributed unequally among the four pancreatic lobes, with most of the A cells located in the third and splenic lobes and PP cells residing in both islet tissue and in acinar tissue. Glucagon appears to be a (the?) major pancreatic hormone involved in metabolic glucoregulation in birds. Yet the essentiality of insulin for this regulatory purpose also has been established. As a result, current thought is directed toward the molar ratio of insulin to glucagon (I/G) as a dominant force in homeostasis rather than toward either of the two hormones separately. Molar I/G ratios have been useful in mammals in studying the needs of the organism to produce glucose to meet a metabolic crisis/need and, when compared with that found in normal Aves, a value of 1.8-2.2 has been established. Such a molar ratio is indicative of a catabolic recovery of nutrients in mammals, suggesting that birds normally are in a catabolic mode (like diabetic, starving, or exercising mammals). Somatostatin (SRIF) is known to inhibit the release of all pancreatic hormones but has a greater inhibitory action on glucagon secretion than it does on any of the other peptides. (It has least effect on APP).(ABSTRACT TRUNCATED AT 250 WORDS)