Natural Infection of Iris yellow spot virus in Twoscale Saltbush (Atriplex micrantha) Growing in Utah.

Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) continues to be an economically important pathogen affecting onion bulb and seed production in several parts of the world and the United States (1). Several weeds were reported naturally infected with IYSV (1,2,4). Leaves of Atriplex micrantha Ledeb. (synonym A. heterosperma Bunge) were collected from naturally occurring plants in a weed trial conducted in commercial onions grown in Box Elder County, UT on 24 September 2008. Leaves displayed a range of symptoms including spotting, chlorosis, and necrosis. Symptomatic leaves were preferentially selected for subsequent diagnostic analyses. Samples were positive for IYSV when tested by double-antibody sandwich-ELISA using a commercially available kit (Agdia Inc., Elkhart, IN). For further confirmation, total nucleic acid extracts from the symptomatic parts of the leaves were prepared and tested for the presence of IYSV by reverse transcription-PCR with primers specific to the nucleocapsid (N) gene coded by the small (S)-RNA of IYSV. The forward and reverse primer pair, 5'-TCAGAAATCGAGAAACTT-3' and 5'-CACCAATGTCTTCAACAATCTT-3', respectively, amplifies a 751-nt fragment of the N gene (3). An amplicon of expected size was obtained, cloned, and sequenced. The nucleotide sequence analysis and comparison with known IYSV S-RNA sequences showed that the sequence of the amplicon from A. micrantha (GenBank Accession No. FJ493541) shared more than 84% nt sequence identity with the corresponding region of IYSV isolates available in GenBank, confirming the IYSV infection of the new host weed. The highest sequence identity (98%) was with an IYSV isolate from Jefferson County, OR (GenBank Accession No. DQ233479). To our knowledge, this is the first report of IYSV infection of A. micrantha under natural conditions. The role of A. micrantha and other weeds in IYSV epidemiology needs further investigation. References: (1) D. Gent et al. Plant Dis. 90:1468, 2006. (2) C. Nischwitz et al. Plant Dis. 91:1518, 2007. (3) H. R. Pappu et al. Arch. Virol. 151:1015, 2006. (4) R. Sampangi et al. Plant Dis. 91:1683, 2007.

Autocrine growth of murine lymphoma cells.

By using an assay system in which small numbers of murine T lymphoma cells are stimulated to grow in serum-free medium, we have continued and expanded our previous studies of an autocrine growth factor that we call leukemia-derived growth factor (LDGF). We show that a T lymphoma cell line of immature phenotype, adapted to growth in serum-free medium, produces and responds to LDGF. LDGF activity is distinct from activities of 10 highly purified or recombinant hematopoietic growth factors including IL-1 and IL-2. However, growth-stimulating activity for the murine lymphoma cells is provided by a partially purified human LDGF.

Identification of serotonin from rabbit upper stomach as a stimulant of in vitro gallbladder contraction.

Using an in vitro rabbit gallbladder bioassay, the distribution and identification of bioactive substances in rabbit gastrointestinal tract were investigated. Comparison of the bioactivities of tissue extracts before and after cholecystokinin was removed by affinity chromatography demonstrated that the distributions of cholecystokinin and non-cholecystokinin substances were different. While cholecystokinin bioactivity per g of tissue was highest in the duodenum, non-cholecystokinin bioactivity was greatest in the upper stomach. The biochemical properties of the non-cholecystokinin substance in the upper stomach could not be distinguished from those of serotonin. These included molecular weights of 176, identical ultraviolet spectra, similar nuclear magnetic resonance spectra, and co-chromatography in HPLC. By weight, serotonin had 1/6th of the bioactivity of cholecystokinin octapeptide. We conclude that the principal gallbladder-contracting substance in rabbit upper stomach is serotonin.

Identification of galanin receptor 1 on excitatory motor neurons in the guinea pig ileum.

Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL-R1). We investigated the effect of exogenous galanin 1-16, which has high affinity for GAL-R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three-compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1-16 (0.3-3 micromol L(-1)) applied to the oral compartment inhibited in a concentration-dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL-R1 antagonist, RWJ-57408 (1 and 10 micromol L(-1)). By contrast, galanin 1-16 was ineffective when added to the intermediate or caudal compartment up to 3 micromol L(-1). GAL-R1 immunoreactive neurons did not contain neuron-specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL-R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons.

Treatment with the ghrelin-O-acyltransferase (GOAT) inhibitor GO-CoA-Tat reduces food intake by reducing meal frequency in rats.

The ghrelin acylating enzyme ghrelin-O-acyltransferase (GOAT) was recently identified and implicated in several biological functions. However, the effects on food intake warrant further investigation. While several genetic GOAT mouse models showed normal food intake, acute blockade using a GOAT inhibitor resulted in reduced food intake. The underlying food intake microstructure remains to be established. In the present study we used an automated feeding monitoring system to assess food intake and the food intake microstructure. First, we validated the basal food intake and feeding behavior in rats using the automated monitoring system. Afterwards, we assessed the food intake microstructure following intraperitoneal injection of the GOAT inhibitor, GO-CoA-Tat (32, 96 and 288 µg/kg) in freely fed male Sprague-Dawley rats. Rats showed a rapid habituation to the automated food intake monitoring system and food intake levels were similar compared to manual monitoring (P = 0.43). Rats housed under these conditions showed a physiological behavioral satiety sequence. Injection of the GOAT inhibitor resulted in a dose-dependent reduction of food intake with a maximum effect observed after 96 mg/kg (-27%, P = 0.03) compared to vehicle. This effect was delayed in onset as the first meal was not altered and lasted for a period of 2 h. Analysis of the food intake microstructure showed that the anorexigenic effect was due to a reduction of meal frequency (-15%, P = 0.04), whereas meal size (P = 0.29) was not altered compared to vehicle. In summary, pharmacological blockade of GOAT reduces dark phase food intake by an increase of satiety while satiation is not affected.

Pancreatic polypeptide from rat pancreas.

Pancreatic polypeptide (PP) has been isolated from rat pancreas by gel filtration, ion exchange chromatography, and high performance liquid chromatography. The isolation was monitored with a RIA, using antibody to the carboxyl-terminal hexapeptide of bovine PP. Rat PP contains 36 amino acids and is similar in composition to PP from other mammalian sources. The single methionine residue in the peptide appears to oxidize easily to the sulfoxide, thereby giving rise to two immunoactive peaks on high performance liquid chromatography. Reduction to the native peptide can be accomplished with mercaptoethanol. The PP content of rat pancreas is about 2 mg/kg. The amino acid sequence of rat PP is Ala-Pro-Leu-Glu-Pro-Met-Tyr-Pro-Gly-Asp- Tyr-Ala-Thr-His-Glu-Gln-Arg-Ala-Gln-Tyr-Glu-Thr-Gln-Leu-Arg-Arg-Tyr-Ile- Asn-Thr-Leu-Thr-Arg-Pro-Arg-Tyr-NH2. This sequence preserves characteristics necessary for stabilization of the compact globular conformation found in avian PP.

Distribution and localization of a novel cholecystokinin-releasing factor in the rat gastrointestinal tract.

The purpose of this study was to examine the distribution and localization of an intestinal cholecystokinin (CCK)-releasing factor, called luminal CCK-releasing factor (LCRF), in the gastrointestinal tract and pancreas of the rat. RIA analysis indicates that LCRF immunoreactivity is found throughout the gut including the pancreas, stomach, duodenum, jejunum, ileum, and colon with the highest levels in the small intestine. Immunohistochemistry analysis shows LCRF immunoreactivity staining in intestinal villi, Brunner's glands of the duodenum, the duodenal myenteric plexus, gastric pits, pancreatic ductules, and pancreatic islets. These results indicate potential sources for secretagogue-stimulated release of luminal LCRF and support the hypothesis that LCRF is secreted into the intestinal lumen to stimulate CCK release from mucosal CCK cells.

The isolation and sequence analysis of vasoactive intestinal peptide from a ganglioneuroblastoma.

A ganglioneuroblastoma was excised at surgery from a 1-yr-old girl with severe watery diarrhea. The tumor, weighing 1 g, was extracted in trifluoracetic acid and contained 8.3 nmol immunoreactive vasoactive intestinal peptide. The peptide was isolated by affinity chromatography and high pressure liquid chromatography and was found to be identical to porcine vasoactive intestinal peptide by amino acid analysis and microsequence analysis.

PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats.

1. Microinjection of peptide YY (PYY, 7-46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY. 2. [Pro34]rat(r)/porcine(p)PYY and [Pro34]human(h)PYY (23-117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68+/-11 and 89+/-31 micromol 90 min(-1) respectively. 3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27+/-8 and 23+/-8 micromol 90 min(-1) respectively) and a higher dose (116 pmol) tended to reduce the response. 4. Pancreatic polypeptide (PP, 4-46 pmol), [Leu31,Pro34]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY3-36, pNPY5-36 and PNPY13-36 (25-168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion. 5. The rank order of potency of PYY > or = [Pro34]r/pPYY = [Pro34]hPYY> r/hNPY = pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu31,Pro34]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.

Reversal by NPY, PYY and 3-36 molecular forms of NPY and PYY of intracisternal CRF-induced inhibition of gastric acid secretion in rats.

1. The Y receptor subtype involved in the antagonism by neuropeptide Y (NPY) of intracisternal corticotropin-releasing factor (CRF)-induced inhibition of gastric acid secretion was studied in urethane-anaesthetized rats by use of peptides with various selectivity for Y1, Y2 and Y3 subtypes: NPY, a Y1, Y2 and Y3 agonist, peptide YY (PYY), a Y1 and Y2 agonist, [Leu31, Pro34]-NPY, a Y1 and Y3 agonist, NPY(3-36) and PYY(3-36), highly selective Y2 agonists and NPY(13-36) a weak Y2 and Y3 agonist. Peptides were injected intracisternally 10 min before intracisternal injection of CRF (10 micrograms) and gastric acid secretion was measured by the flushed technique for 1 h before and 2 h after pentagastrin-(10 micrograms kg-1 h-1, i.v.) infusion which started 10 min after CRF injection. 2. Intracisternal injection of CRF (10 micrograms) inhibited by 56% gastric acid secretion stimulated by pentagastrin. Intracisternal injection of NPY and PYY (0.1-0.5 microgram) did not influence the acid response to pentagastrin but blocked CRF-induced inhibition of pentagastrin-stimulated acid secretion. NPY(3-36) (0.5 microgram) and PYY(3-36) (0.25 and 0.5 microgram) also completely blocked the inhibitory action of CRF on pentagastrin-stimulated acid secretion. 3. [Leu31, Pro34]-NPY (0.5-5 micrograms) and NPY(13-36) (0.5-5 micrograms) injected intracisternally did not modify gastric acid secretion induced by pentagastrin or CRF inhibitory action. 4. The sigma antagonist, BMY 14802 (1 mg kg-1, s.c.) did not influence the acid response to pentagastrin but prevented the antagonism by PYY(3-36) (0.5 microgram) of the CRF antisecretory effect. 5. These results show that both PYY and NPY and the 3-36 forms of PYY and NPY are equipotent in blocking central CRF-induced inhibition of pentagastrin-stimulated gastric acid secretion. The structure-activity profile suggests a mediation through Y2 receptor subtype and the involvement of sigma binding sites.

Processing of mammalian preprogastrin-releasing peptide.

The processing of preprogastrin-releasing peptide in mammalian tissues and in cultured cells takes place at discrete sites (Figure 6). Signal peptidase cleaves away the signal peptide from the amino terminus of gastrin-releasing peptide. An exopeptidase activity may remove dipeptides from the amino terminus. The amidation site (not shown in Fig. 6; see Fig. 2) has the same general sequence (Gly-Lys-Lys) seen for other amidated peptides. Cleavage after single basic residues yields gene-related products from Form I or II preproGRP. A unique non-basic cleavage yields a gene-related product from Form III preproGRP. The processing that occurs to form GRP, GRP, and GRP gene-related peptides is shown in Figure 7. ProGRP is cleaved by a series of enzymes to form GRP with an amidated carboxyl-terminal methionine (indicated by an asterisk in Fig. 7). GRP is cleaved to form the decapeptide GRP. The carboxyl-terminal flanking peptides of all three mRNA translation products are cleaved to form several gastrin-releasing peptide gene-related products. Knowledge of the processing of gastrin-releasing peptide and its gene-related products will allow synthesis of duplicates of the stored forms of these peptides, which can then be used for biological testing.

Natural and synthetic CCK-58. Novel reagents for studying cholecystokinin physiology.

CCK-58 is a unique reagent for testing how segments of a peptide far removed from its active site can influence the expression of its biological activity. Indications of tertiary structure have come from studies with natural peptide purified from canine small intestine. These studies gave clear indications that tertiary structure affects CCK-58 bioactivity, but the small quantities of CCK-58 available made it impossible to characterize completely how tertiary structure influenced bioactivity. Canine CCK-58 was synthesized manually using a solid support and was purified by reverse phase high pressure liquid chromatography (HPLC). Synthetic CCK-58 was characterized by isocratic reverse phase and gradient HPLC, amino acid analysis, mass spectral analysis, sequence analysis, and three bioassays. Synthetic and natural canine CCK-58 had the same elution profiles, amino acid composition, sequence, and mass. The two peptides were equipotent for the stimulation of pancreatic secretion. Natural canine CCK-58 was equipotent to CCK-8 for CCK "B" receptor binding, a further indication of the purity of the natural peptide. However, natural CCK-58 was more potent than CCK-8 for CCK "A" receptor binding and less potent than CCK-8 for stimulation of pancreatic secretion. These data support the concept that CCK-58 has a stable tertiary structure. This structure does not affect its binding to CCK "B" receptors, enhances its binding to low affinity CCK "A" receptors, and decreases its activity expressed through binding to high affinity CCK "A" receptors. The concept of a stable tertiary structure is also supported by the fact that many antibodies directed towards the carboxyl terminus of cholecystokinin react better with CCK-8 than CCK-58. The availability of synthetic CCK-58 will allow analysis of its tertiary structure by physical and chemical methods as well as studies on how peptide tertiary structure can affect receptor binding, receptor activation, metabolism in blood, degradation in interstitial fluid, and inactivation at the receptor. Evaluating all of these systems will help investigators understand the regulation of cholecystokinin activity by its major endocrine form, CCK-58.

CCK-58: a novel reagent for studying the regulation of cholecystokinin bioactivity.

CCK-58 has been shown to be the major circulating form of the hormone in the dog and human. To date, there have been no reports on its biological activity in vivo. We report here that CCK-8 and CCK-58 were equipotent in decreasing gastric motor function after bolus doses and in stimulating protein secretion after continuous infusion in urethane-anesthetized rats. The present results are the first on the in vivo activity of CCK-58, and indicate that because CCK-58 is equipotent to CCK-8, and because it is a major released and circulating form, it may be considered as a major contributor to the expression of cholecystokinin bioactivity.

Mammalian bombesin-like peptides: neuromodulators of gastric function and autocrine regulators of lung cancer growth.

Peptides corresponding closely in structure to the biologically active carboxyl terminal region of the amphibian peptide bombesin have now been isolated from several mammalian species, including man. Two principal forms have been found: one contains 27 amino acids and exhibits variations in amino acid sequence in the amino terminal region; the other is the carboxyl terminal decapeptide and probably does not vary among mammals. These peptides exhibit full immunoreactivity with most bombesin antisera and account for "bombesin-like immunoreactivity" that has been described in mammalian brain, sympathetic ganglia, and nerve fibers in the gut as well as in fetal lung endocrine cells and certain lung tumors, especially small cell lung carcinoma. The name gastrin releasing peptide (GRP) was given to the porcine and avian heptacosapeptides by McDonald and Mutt. The larger and smaller mammalian peptides now often are called GRP27 and GRP10. Both forms exhibit the full spectrum of activity shown by bombesin. Evidence has been obtained that neural release of mammalian bombesin-like peptides is physiologically important in regulation of gastrin release from the stomach. Lung tumors that produce bombesin-like peptides also have receptors for bombesin. These receptors appear to be involved in the autocrine regulation of tumor cell proliferation.

Relative bioactivities of cholecystokinins-8 and -33 on rat pancreatic acini.

The relative potencies of cholecystokinin (CCK-33) and its carboxyl terminal octapeptide (CCK-8) for stimulation of amylase release from rat pancreatic acini was measured. Porcine CCK-33 and synthetic CCK-8 were initially subjected to high pressure liquid chromatography to assess purity. Concentrations of each peptide were determined by amino acid analysis. The relative immunoreactivities of CCK-33 and CCK-8 were compared using an antibody that recognizes the common carboxyl terminus of these forms. This antibody bound CCK-8 and CCK-33 with nearly equal affinity. The relative potencies of CCK-33 and CCK-8 were then measured by comparing their abilities to stimulate amylase release from isolated rat pancreatic acini. Statistical analysis of the relative potencies of the two hormones indicated that CCK-8 was 36% more potent than CCK-33 in this assay system. These data suggest that differences in biological activities between large and small forms of CCK are not as great as previously reported.

Isolation and characterization of rat vasoactive intestinal peptide.

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.

Isolation of the neuropeptide SALMFamide-1 from starfish using a new antiserum.

We have raised antisera in rabbits to a conjugate of thyroglobulin and Lys-Tyr-Ser-Ala-Leu-Met-Phe-NH2 (KYSALMFamide), a synthetic analog of the starfish neuropeptide S1 (Gly-Phe-Asn-Ser-Ala-Leu-Met- Phe-NH2). The sensitivity and specificity of two antisera (BL and SL) for S1 were established by testing the ability of S1 and structurally related peptides (SALMFamide-2 and various FMRFamide-related peptides) to displace iodinated KYSALMFamide from the serum antibodies in an RIA. Both antisera are sensitive to femtomolar amounts of S1. BL is highly specific for S1 but SL is not, since it is also able to detect femtomolar amounts of the FMRFamide-related peptides. We have used the BL antiserum in the RIA to monitor the purification of S1 immunoreactivity from radial nerve cord extracts of both Asterias rubens and Pycnopodia helianthoides. The partial amino acid sequence GFNSALM was obtained from automated Edman degradation sequencing of pure immunoreactive peaks from both species.

Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.

The development of a biotinylated bombesin/gastrin-releasing peptide (GRP) for use as a receptor probe is reported. The lysine13 of a GRP-27 was substituted by arginine and lysine was added to the amino terminus. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by HPLC and characterized by mass spectral analysis. Binding studies with murine Swiss 3T3 fibroblasts, cells known to express bombesin/GRP receptors, yielded a dissociation curve for the biotinylated GRP-27 analogue (biotin-Lysyl[Asp12,Arg13]GRP-27) which was nearly identical to that of native GRP. Using studies of gastrin release from isolated canine G cells, equipotent functional activity of the biotinylated probe and unmodified GRP was demonstrated. Measurements of retained 125I-avidin confirmed that the biotin/avidin interaction could occur once the biotin-peptide complex was bound. Applicability of the probe was demonstrated with fluorescent microscopy using avidin-FITC on Swiss 3T3 fibroblasts. In conclusion, a novel biotinylated bombesin/GRP analogue has been developed which retains the functional characteristics of the native peptide and is a useful probe for receptor studies.

Unique amino terminal structure of rat little gastrin.

The heptadecapeptide form of rat gastrin was purified by a combination of DEAE cellulose, Sephadex G50 affinity, and high performance liquid chromatography. An amino terminal pyroglutamyl blocking group was removed by incubation with PCA peptidase. Amino acid analysis before and after the unblocking reaction revealed the presence of one additional residue of arginine and proline compared with porcine gastrin. Microsequencing analysis of the unblocked peptide revealed that the sequence of the remaining hexadecapeptide was RPPMEEEEEAYGWMDF. The corresponding sequence of porcine gastrin is GPWMEEEEEAYGWMDF amide. The presence of carboxyl-terminal amide group in rat gastrin is strongly supported by complete immunoreactivity with antibodies specific for amidated C-terminal sequences of mammalian gastrins. The Arg and Pro substitutions in the amino terminal region can explain poor crossreactivity of rat gastrin with antibodies specific for the amino-terminal portion of porcine or human gastrin and its more basic chromatography pattern on ion exchange resins.

Characterization of two forms of peptide YY, PYY(1-36) and PYY(3-36), in the rabbit.

Peptide YY (PYY) has been purified as a 36 amino acid peptide from intestinal extracts of several mammalian species including pig, rat, dog, and man. The primary structure of rabbit PYY is still unknown, although rabbit tissues have extensively been used for characterization of PYY receptor subtypes and receptor subtype-mediated actions. We report the purification and primary structure of PYY(1-36) (PYY-I) from rabbit intestinal mucosa, and the existence of a second endogenous molecular form of PYY, PYY(3-36) (PYY-II). The amino acid sequence of PYY-I is YPSKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide. Rabbit PYY differs from porcine PYY, which is identical to rat and canine PYY, by two amino acid substitutions at positions 3 (Ser instead of Ala) and 18 (Asp instead of Ser), whereas rabbit PYY and human PYY differ by only one residue at position 3 (Ser instead of Ile). The existence of two endogenous forms of PYY in the rabbit, with PYY-II lacking the amino-terminal dipeptide Tyr-Pro of PYY-I, is consistent with previously reported findings, demonstrating the existence of PYY-II in man and dog (9,11). We have previously demonstrated that PYY-I is an unselective Y1/Y2 agonist, whereas PYY-II is a highly selective Y2 agonist. Thus, proteolytic processing of PYY-I controls the peptide's receptor selectivity. The existence of PYY-I and PYY-II in the rabbit supports the assumption of a physiological role of Y receptor heterogeneity for PYY.