Cholecystokinin-like Peptides Research Articles

Drosulfakinin signaling modulates female sexual receptivity in Drosophila

Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. Here, we identify the Cholecystokinin-like peptide Drosulfakinin (DSK) to promote female sexual behavior in Drosophila. Loss of DSK function reduces female receptivity while overexpressing DSK enhances female receptivity. We identify two pairs of Dsk-expressing neurons in the central brain to promote female receptivity. We find that the DSK peptide acts through one of its receptors, CCKLR-17D3, to modulate female receptivity. Manipulation of CCKLR-17D3 and its expressing neurons alters female receptivity. We further reveal that the two pairs of Dsk-expressing neurons receive input signal from pC1 neurons that integrate sex-related cues and mating status. These results demonstrate how a neuropeptide pathway interacts with a central neural node in the female sex circuitry to modulate sexual receptivity.

Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction.

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. Here, we investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. We found that feeding increases the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, we found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, we have identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. Our findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but our experiments cannot exclude further parallel pathways. Importantly, we show that the underlying mechanisms are conserved in the two distantly related insect species.

Insulin-Like Peptides and Cross-Talk With Other Factors in the Regulation of Insect Metabolism

The insulin-like peptide (ILP) and insulin-like growth factor (IGF) signalling pathways play a crucial role in the regulation of metabolism, growth and development, fecundity, stress resistance, and lifespan. ILPs are encoded by multigene families that are expressed in nervous and non-nervous organs, including the midgut, salivary glands, and fat body, in a tissue- and stage-specific manner. Thus, more multidirectional and more complex control of insect metabolism can occur. ILPs are not the only factors that regulate metabolism. ILPs interact in many cross-talk interactions of different factors, for example, hormones (peptide and nonpeptide), neurotransmitters and growth factors. These interactions are observed at different levels, and three interactions appear to be the most prominent/significant: (1) coinfluence of ILPs and other factors on the same target cells, (2) influence of ILPs on synthesis/secretion of other factors regulating metabolism, and (3) regulation of activity of cells producing/secreting ILPs by various factors. For example, brain insulin-producing cells co-express sulfakinins (SKs), which are cholecystokinin-like peptides, another key regulator of metabolism, and express receptors for tachykinin-related peptides, the next peptide hormones involved in the control of metabolism. It was also shown that ILPs in Drosophila melanogaster can directly and indirectly regulate AKH. This review presents an overview of the regulatory role of insulin-like peptides in insect metabolism and how these factors interact with other players involved in its regulation.

Drosulfakinin signaling in fruitless circuitry antagonizes P1 neurons to regulate sexual arousal in Drosophila

Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. Here, we identify the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. We found that Dsk neurons physiologically interact with male-specific P1 neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. We further found that the DSK-2 peptide functions through its receptor CCKLR-17D3 to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless-expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P1 neurons to balance arousal levels and modulate sexual behaviors.

Insulin-Producing Cells in the Drosophila Brain also Express Satiety-Inducing Cholecystokinin-Like Peptide, Drosulfakinin

Regulation of meal size and assessing the nutritional value of food are two important aspects of feeding behavior. The mechanisms that regulate these two aspects have not been fully elucidated in Drosophila. Diminished signaling with insulin-like peptides Drosophila insulin-like peptides (DILPs) affects food intake in flies, but it is not clear what signal(s) mediates satiety. Here we investigate the role of DILPs and drosulfakinins (DSKs), cholecystokinin-like peptides, as satiety signals in Drosophila. We show that DSKs and DILPs are co-expressed in insulin-producing cells (IPCs) of the brain. Next we analyzed the effects of diminishing DSKs or DILPs employing the Gal4-UAS system by (1) diminishing DSK-levels without directly affecting DILP levels by targeted Dsk-RNAi, either in all DSK-producing cells (DPCs) or only in the IPCs or (2) expressing a hyperpolarizing potassium channel to inactivate either all the DPCs or only the IPCs, affecting release of both peptides. The transgenic flies were assayed for feeding and food choice, resistance to starvation, and for levels of Dilp and Dsk transcripts in brains of fed and starved animals. Diminishment of DSK in the IPCs alone is sufficient to cause defective regulation of food intake and food choice, indicating that DSK functions as a hormonal satiety signal in Drosophila. Quantification of Dsk and Dilp transcript levels reveals that knockdown of either peptide type affects the transcript levels of the other, suggesting a possible feedback regulation between the two signaling pathways. In summary, DSK and DILPs released from the IPCs regulate feeding, food choice and metabolic homeostasis in Drosophila in a coordinated fashion.

Fluorescence Polarization Screening for Allosteric Small Molecule Ligands of the Cholecystokinin Receptor

The success in screening for drug candidates is highly dependent on the power of the strategy implemented. In this work, we report and characterize a novel fluorescent benzodiazepine antagonist of the type 1 cholecystokinin receptor (3-(3-(7-fluoro-1-(2-isopropyl(4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]-diazepin-3-yl)ureido)benzoic acid) that can be used as a receptor ligand in a fluorescence polarization assay, which is ideally suited for the identification of small molecule allosteric modulators of this physiologically important receptor. By binding directly to the small molecule-docking region within the helical bundle of this receptor, this indicator can be displaced by many small molecule candidate drugs, even those that might not affect the binding of an orthosteric cholecystokinin-like peptide ligand. The biological, pharmacological, and fluorescence properties of this reagent are described, and proof-of-concept is provided in a fluorescence polarization assay utilizing this fluorescent benzodiazepine ligand.

Luminal Cholecystokinin and Gastrin Cause Gallbladder Contraction

Cholecystokinin-8 placed in the gallbladder lumen causes gallbladder contraction by a neurally mediated, tetrodotoxin-sensitive mechanism. We wished to determine whether other cholecystokinin-like peptides in the gallbladder lumen cause contraction and whether this response is inhibited by cholecystokinin-receptor antagonists. In this study, we measured gallbladder contraction induced by cholecystokinin-like peptides or by hepatic bile placed in the gallbladder lumen. Isolated gallbladders were suspended in an organ bath while luminal hormones were infused. Gallbladder contraction was measured by continuous monitoring of luminal pressure. Cholecystokinin-8, cholecystokinin-5, and gastrin-17 caused dose-related gallbladder contraction with similar potency when placed in the lumen. Each stimulated 70–80% maximal contraction at a luminal concentration of 10−6M.Cholecystokinin-receptor antagonists CR1409 and loxi- glumide partially inhibited contraction caused by luminal cholecystokinin-8. Bile from fed animals, but not from fasted animals, stimulated gallbladder contraction to 36 ± 4% of maximal when placed in the gallbladder lumen. We conclude that cholecystokinin and gastrin peptides in the gallbladder lumen cause contraction. This may be mediated through receptors of the cholecystokinin-B type, possibly on intrinsic nerves. Bile from fed animals also contains substances that stimulate gallbladder contraction when bile is placed in the gallbladder lumen. These findings suggest intrinsic nerves participate in the postprandial gallbladder response to cholecystokinin.

Effects of ceruletide and haloperidol on auditory evoked potentials in the cat brain.

The influence of cholecystokinin-like peptide, ceruletide, on EEG and auditory evoked potentials (AEPs) was studied in nine cats. The cats were bearing electrodes implanted in the auditory cortex, hippocampus, reticular formation and cerebellum. Reference drugs used were haloperidol and neostigmine. The hippocampus showed the strongest effect of ceruletide, whereas the cerebellum was virtually unresponsive. The amplitude of AEPs was increased by peptide, an effect lasting up to 21 days which, according to amplitude frequency analysis (AFC) was due to an augmented theta response. The latter possibly indicates increased signal transfer to, or through, the brain structure in question, particularly in the hippocampal neurons. The effects of haloperidol and neostigmine did not reflect those of ceruletide and lasted only a few hours.

Immunocytochemical Localization Of Multiple Cholecystokinin-Like Peptides In The Stomatogastric Nervous System Of The Crab Cancer Borealis

Three anti-cholecystokinin antibodies were used to label the stomatogastric nervous system of the crab Cancer borealis. Labeled tissues were examined as whole mounts using laser scanning confocal microscopy. Although each of the anti-cholecystokinin antibodies labeled a variety of structures within the stomatogastric nervous system (including somata, fibers and neuropil), the pattern of labeling produced by each antibody was distinct. These results indicate that there is a family of cholecystokinin-like molecules that are differentially distributed among a subpopulation of the neurons in the stomatogastric nervous system of Cancer borealis.

Partial purification, tissue distribution and modulatory activity of a crustacean cholecystokinin-like peptide.

Reversed-phase chromatography was used to separate several forms of cholecystokinin-like peptides (CCKLP) from the pericardial organs (PCOs) of the spiny lobster Panulirus interruptus. Fast protein liquid chromatography of PCOs, stomatogastric ganglia (STGs) and eyestalks revealed five peaks of CCKLP (peaks A-E) that were common to all three tissues, as well as two additional peaks (peaks F and G) in the STG. Peaks A-E were present in the hemolymph of fed, but not starved, lobsters. The bioactivity of peaks A-E was tested on the gastric mill rhythm of the isolated STG. Only peak E elicited activity. The effects of peak E included activating the gastric mill rhythm in quiescent preparations and strengthening existing rhythms in a dose-dependent manner. Further purification of peak E by high performance liquid chromatography resolved this peak into two immunoreactive peaks, one of which retained its bioactivity. The effects of peak E were blocked by the CCK antagonist proglumide. These results are consistent with a role for peak E in the feeding-induced activation of the gastric mill.

Facilitation of defense reactions during the consumption of food in snails: the participation of glucose and gastrin/cholecystokinin-like peptide.

The molecular-cellular mechanisms of the interrelationships between the alimentary and defense behavior of snails were investigated. The clarity of manifestation of defense reactions at various stages of the satisfaction of alimentary need was studied. It was found that the defense reactions were attenuated in starved molluscs, whereas after a 20-minute feeding session the facilitation of generalized defense reactions takes place, the clarity of manifestation of which depends on the duration of food deprivation. It was demonstrated that an increase in the level of glucose in the hemolymph of the starved molluscs to the level present in the hemolymph during a meal (1.6-2.0 mM), as well as the administration of 20-30 ng of analogs of gastrointestinal peptides (pentagastrin or cholecystokinin octapeptide, CCK-8) elicits the facilitation of generalized defense reactions similar to that found during the feeding of the snails. The directionality of the changes in the activity of the L-RPl1 neurons during the application of carrot juice to the lip of semi-intact preparations of starved snails depended on the presence of glucose in the solution bathing the central nervous system. Similar glucose-dependant changes were found after the application of CCK-8 to the CNS of starved snails, but not of FMRFamide, whereas the isolated application of glucose did not exhibit such an effect. It was found that the L-RPl1 neurons were the most sensitive to the change in the concentration of glucose and CCK-8 in the perfusate. The adaptive significance of the dependance of the clarity of manifestation of defense reactions on the level of alimentary need of the snails and the participation of glucose and gastrin or cholecystokinin-like peptide in these processes are discussed.

In man the mu-opiate agonist loperamide specifically inhibits ACTH secretion induced by the cholecystokinin-like peptide ceruletide.

Ceruletide, a cholecystokinin-8-like peptide, was recently reported to stimulate ACTH secretion in man. The aim of this study was to investigate the effect of the mu-opiate agonist, loperamide, and the opiate antagonist, naloxone, on ceruletide-induced ACTH secretion in man. In 6 normal subjects, basal ACTH and cortisol plasma levels were significantly suppressed 3 h after loperamide administration (16 mg, orally) from 5 +/- 0 to 2 +/- 0 pmol/l and from 356 +/- 44 to 154 +/- 16 nmol/l. After stimulation with 8 ng ceruletide/kg body weight/min over a period of 5 min, the maximum ACTH levels (at 7.5 min) were significantly reduced by loperamide from 26 +/- 7 to 6 +/- 1 pmol/l and the maximum cortisol levels (at 30 min) were significantly reduced from 676 +/- 47 to 392 +/- 58 nmol/l. Furthermore, the ACTH peak (delta = 7.5 min) was significantly blunted by loperamide from 21 +/- 7 to 5 +/- 1 pmol/l and the integrated area under the curve from 0 to 120 min (delta AUC) of ACTH was significantly reduced from 40 +/- 11 to 14 +/- 4 pmol x 120 min/l. The cortisol peak (delta = 30 min) and the AUC of cortisol were not significantly diminished. The suppressive effect of loperamide on basal and ceruletide-induced ACTH and cortisol secretion was completely reversed by the administration of 0.8 mg naloxone, 20 min before and during infusion of ceruletide. The administration of naloxone itself had no significant effect on ACTH or cortisol levels. In conclusion, ACTH is released by peripherally administered ceruletide within a short period.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of cholecystokinin-like and calcitonin-like peptides in infant carotid bodies: a light- and electron-microscopic immunohistochemical study

Previous immunohistochemical studies have identified several regulatory peptides in the carotid body chief cells in both humans and animals. These peptides, together with amines, may be important in the modulation of the chemoreflex by the carotid body. We report the localization and distribution of calcitonin and cholecystokinin-like (CCK) immunoreactivity in chief cells of human infant carotid body by light- and electron-microscopic immunohistochemical techniques. Consecutive sections immunostained with calcitonin and/or CCK antibodies revealed positively stained chief cells, both alone and in clusters, scattered throughout the carotid body lobule. Generally more chief cells were positive for calcitonin than for CCK. This was confirmed by quantitative analysis showing that the ratio of calcitonin to CCK immunoreactive cells was consistently > 2:1 in all cases studied. There was no apparent correlation between the immunoreactivity for the two peptides and the age, sex, or postmortem interval. Calcitonin-like and CCK-like immunoreactivities were localized electron-microscopically over the dense core granules of the chief cells. Calcitonin and CCK-like peptides in carotid body chief cells may act as neurotransmitters or neuromodulators involved in chemoreception.

Ceruletide, a CCK-like peptide, attenuates dopamine release from the rat striatum via a central site of action

Ceruletide (CLT), a cholecystokinin-like peptide was given subcutaneously or via the perfusate to rats to clarify the site of action (peripheral vs. central location) of CLT, using in vivo microdialysis techniques. Striatal dopamine (DA) release induced by haloperidol (HPD) was significantly inhibited by subcutaneously administered CLT (160 μg/kg) when given with a perfusate containing 15 mM K +. Subdiaphragmatic vagotomies failed to block the inhibitory effect of CLT. CLT (10 −15−10 −11 M) locally applied, via a dialysis tube, produced an inhibitory effect on HPD-induced DA release in the striatum in a dose-dependent manner. The inhibitory effect of CLT given subcutaneously on DA release was antagonized by both locally applied proglumide and systemically administered L-365,260. These findings suggest that systemically administered CLT can directly act on the striatal neurons via CCK-B receptors and produce an inhibitory effect on DA release in the striatum under appropriate depolarization.

A cholecystokinin-like hormone activates a feeding-related neural circuit in lobster

The peptide hormone cholecystokinin (CCK) contributes to the production of feeding-related behaviour in mammals, but the mechanism by which it exerts its effects remains unclear. The gastric mill neural circuit of lobster is an experimentally accessible model system for studying the hormonal control of feeding-related behaviour. Composed of 11 identified neurons, this circuit produces rhythmic movement of teeth within the stomach. We have previously shown that the gastric mill motor pattern can be modulated by a cholecystokinin-like peptide in vitro. We report here that (1) after feeding, levels of CCK-like peptide in haemolymph increase with the activation of the gastric mill, (2) injections of CCK activate the gastric mill, and (3) a specific CCK antagonist inhibits feeding-induced gastric mill activity. This neatly demonstrates a casual link between in vivo release of a peptide hormone and activation of a neural circuit.

Interaction of CCK with pancreatic acinar cells

Recent studies demonstrate that cholecystokinin-like peptides are widely distributed in the CNS as well as in the peripheral nervous system and gastrointestinal tract. Studies with agonists have demonstrated multiple classes of receptors and recently potent receptor antagonists have been described which will distinguish these classes and should allow a better understanding of the role of CCK in various physiological processes. One of the known peripheral physiological functions of CCK is the stimulation of digestive enzymes from pancreatic acinar cells. In recent years the interaction of CCK with pancreatic acinar cells has been extensively studied and significant advances have been made in understanding its cellular basis of action. Robert Jensen and colleagues report on each of these areas.

Cholecystokinin-like peptide is a modulator of a crustacean central pattern generator

The presence, release, and physiological effects of a cholecystokinin(CCK)-like peptide within the stomatogastric ganglion (STG) of the lobster, Panulirus interruptus, are described. Indirect immunofluorescence with 2 antisera raised against CCK8 was used to determine the distribution of CCK-like immunoreactivity (CCKLI) in the stomatogastric nervous system. CCKLI was demonstrated in the input nerve and the neuropil of the STG and in neuropil and somata in the commissural ganglia (CGs), brain, and eyestalks. None of the somata within the STG displayed CCKLI. The cross-reactivities of the CCK antisera with several peptides were determined using either a radioimmunoassay or an immunoblot assay; the antisera recognized peptides homologous to CCK but did not cross-react significantly with several unrelated peptides. The STG contains 2 central pattern generators (CPGs), the pyloric and the gastric mill CPGs. Bath application of CCK8 to the STG had modulatory effects on both CPGs, which were dose dependent and reversible. CCK increased the spike frequencies and number of spikes per burst of the pyloric rhythm but had little effect on the period. CCK increased the period of the gastric rhythm and produced changes in the spike frequencies, burst lengths, and phases of gastric units. High concentrations of peptide were needed to produce these effects (10(-6) to 10(-4) M). Finally, stimulation of the stomatogastric nerve (stn), which contains fibers immunoreactive to CCK, produced calcium-dependent release of CCK molar equivalents (CCKE) into the STG. The stn was electrically stimulated and the superfusate around the ganglion was collected and assayed for CCKE using a radioimmunoassay. Stimulation produced the release of 37.1 +/- 7.1 fmol (mean +/- SEM), compared to 13.7 +/- 4.9 fmol for unstimulated controls and 4.9 +/- 2.9 fmol in the absence of calcium. These data suggest that a CCK-like peptide is an endogenous modulator of the stomatogastric ganglion of P. interruptus.

Short- and Long-Term Effects of Secretin and a Cholecystokinin-Like Peptide on Pancreatic Growth and Synthesis of RNA and Polyamines

Little is known about the cellular mechanisms responsible for the trophic effects of cholecystokinin (CCK) and secretin on the rat pancreas, and controversy exists with regard to the interaction between these two peptides. In the present study attempts were made to elucidate the time course of events leading to pancreatic growth and to clarify the interaction between the peptides when given as continuous, long-term intravenous infusions to rats. A cholecystokinin-like peptide (CCK-LP) and secretin were given as a continuous intravenous infusion to conscious and unrestrained animals with free access to food and water for 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 96 h. The pancreas was quickly removed and analyzed for variables indicating synthesis and accumulation of DNA, RNA, and polyamines. CCK-LP increased the activity of RNA polymerase already after 1 h, whereas an increase in the activity of ornithine decarboxylase (ODC) and the level of putrescine was seen at 4 h. Spermidine was increased after 12 h. The activities of DNA polymerase and thymidine kinase were increased at 12 and 24 h, respectively, whereas the total contents of DNA and RNA were first increased at 48 h. Secretin alone showed a marked but short-lived effect on polyamine synthesis and a weak effect on the variables indicating protein synthesis and growth. When the two peptides were given together, a large but transient potentiation of ODC activity was observed, whereas no interaction was seen on polyamines, RNA synthesis, or pancreatic growth. The present study confirms the trophic effects of CCK and secretin on the rat pancreas but fails to confirm an interaction between the two peptides on growth. Both peptides stimulate polyamine synthesis, and ODC appears to be an early and sensitive indication of their trophic effect. The initiation of RNA synthesis appears to be independent of the ODC activity.

Evidence for an involvement of substance P, but not cholecystokinin-like peptides, in hexamethonium-resistant intestinal peristalsis

It has previously been found that, in the presence of naloxone, the ganglionic blocking drug hexamethonium fails to completely block peristaltic motility in the isolated ileum of the guinea-pig. This hexamethonium-resistant peristaltic activity is coordinated by enteric nerves since it is abolished by tetrodotoxin. In the present study the neurotransmitter circuitry of this type of peristalsis was studied by means of specific antagonists. Atropine totally suppressed hexamethonium-resistant peristalsis. This type of peristalsis was also strongly inhibited by the tachykinin antagonist, spantide, if a concentration sufficient to antagonize neuronally located substance P receptors was employed. In contrast, the cholecystokinin antagonist, lorglumide, caused only a slight inhibition of hexamethonium-resistant peristalsis. Both substance P and the cholecystokinin-related peptide, ceruletide, potently stimulated the hexamethoniumresistant type of peristaltic activity. These data indicate that, after blockade of nicotinic acetylcholine receptors, tachykinins mediate neuroneuronal coordination of peristalsis whereas acetylcholine acting via muscarinic receptors may be primarily responsible for neuromuscular transmission. Cholecystokinin-like peptides appear to play a modulator rather than a mediator role in hexamethonium-resistant peristalsis.

The Effects of Graded Doses of a Cholecystokinin-Like Peptide with and without Secretin on Pancreatic Growth and Synthesis of RNA and Polyamines in Rats

The dose dependence of a cholecystokinin-like peptide (CCK-LP) on the trophic response in the rat pancreas was studied. Graded doses of Thr28Nle31CCK25-33 (0.02, 0.1, 0.5, 2.5, and 12.5 micrograms/kg/h) or saline were given as a continuous intravenous infusion to conscious and fed rats for 8 and 48 h. Secretin (5.0 micrograms/h) was given alone or combined with the three highest doses of CCK-LP for 48 h. CCK-LP showed a dose-dependent stimulating effect on pancreatic growth and synthesis of RNA and polyamines. The threshold dose ranged from 0.02 to 0.5 micrograms/kg/h and was lowest for stimulation of ornithine decarboxylase (ODC). The maximal effects on protein, RNA, and DNA contents were achieved with 2.5 micrograms/kg/h. These same variables markedly decreased with 12.5 micrograms/kg/h, whereas marked further increases were found for the activities of RNA polymerase, DNA polymerase, and thymidine kinase. This same dose of CCK-LP caused after 8 h of treatment a marked and transient increase in pancreatic weight, activity of ODC, and concentration of putrescine. When secretin was added to 0.5 and 2.5 micrograms/kg/h of CCK-LP, no additional effect (except for ODC) was found. When secretin was added to the highest dose of CCK-LP, the decreased contents of protein and RNA were significantly increased, and the markedly increased activities of RNA- and DNA-synthesizing enzymes were significantly decreased. The present study shows a clear dose-response relationship for the trophic effect of CCK-LP on the rat pancreas and indicates that the growth effect of a supramaximal dose includes components of regeneration secondary to damage.(ABSTRACT TRUNCATED AT 250 WORDS)