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It has been suggested that convolution functions other than Ernst's optimal matched filter may be efficacious for resolution enhancement of NMR spectra. For the purposes of comparison, the possible line distortions and signal-to-noise and resolution enhancement ratios in the Ernst filter are explicitly derived. The general limitation inherent in the digital transformation on the minimum linewidth obtainable by any convolution filter is also derived.

Levels of delta-sleep-inducing peptide-like immunoreactivity (DSIP-LI) in rat plasma were measured by radioimmunoassay and found to exhibit a circadian rhythm that parallelled the normal rhythm for corticosterone. The maximal plasma levels of both substances were observed to occur at about 1700h. The lowest concentrations of DSIP-LI and corticosterone were detected at 2400h and 1000h, respectively. Exposure to constant levels of illumination abolished the rhythm of DSIP-LI. It is possible that the temporal parallelism between the levels of DSIP-LI and corticosterone may represent a functional relationship between both compounds.

Corticotropin releasing factor (CRF) was recently isolated from ovine hypothalami by its ability to stimulate adrenocorticotropin (ACTH) and β-endorphin release from dispersed rat pituitary cells. Intramuscular injection of synthetic ovine CRF conugated to bovine thyroglobulin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and emulsified with Freund's complete adjuvant into a random bred New Zealand white rabbit resulted in antiserum production to CRF associated with adrenal atrophy. A decrease in the level of plasma coticosteroids was associated with an increase in mean total binding of 125 I-N-Tyr-CRF. Upon sacrifice, a decrease in pituitary content of ACTH and a decrease in adrenal weight and content of corticosteroids was observed in the rabbit producing antiserum to CRF. Adrenal atrophy was histologically verified with an observed decrease in the adrenal cortical zone not reflected in the zona glomerulosa. Individual cells were relatively larger either with more abundant pale cytoplasm or with distinctly vacuolated cytoplasm. The results presented here are consistent with a physiologically necessary role for this CRF or peptides with similar structures in the hypothalamic-pituitary-adrenal axis.

CRF-like immunoreactivity was measured by radioimmunoassay in the brains of normal adult rats and found to be widely distributed in extrahypothalamic areas (e.g., thalamus, amygdala, hippocampus, frontal cerebral cortex, striatum, midbrain, pons-medulla and cerebellum) at levels approximately 10% of the hypothalamus. Sephadex G-50 gel filtration reveals that CRF-like immunoreactivity in the hypothalamus coelutes with synthetic ovine CRF and is also present in the void volume. However, in the extrahypothalamic areas of the rat brain, only CRF-like immunoreactivity that coelutes with synthetic ovine CRF was detected. High performance liquid chromatography revealed equal amounts of immunoreactivity coeluting with CRF and methionine sulfoxide CRF in hypothalamic extracts.

Plasma membrane injury by exposure to hydrogen peroxide was examined in a renal epithelial cell line (LLC-PK1). Morphologic and functional parameters of plasma membrane integrity were studied in an attempt to eludicate the sequence of membrane alterations during the evolution of hydrogen peroxide-mediated injury. These parameters included plasma membrane potential and permeability, plasma membrane bleb formation, cellular size, and plating efficiency. Plasma membrane potential was the earliest parameter affected by hydrogen peroxide exposure. Half maximal depolarization occurred within 15-30 min of exposure to 1 mM, after 10-15 min exposure to 100 mM and after over 150 min exposure to 10 microM hydrogen peroxide. After exposure to 1 mM hydrogen peroxide, the following sequence of events was seen; increased plasma membrane blebbing (30 min), cell swelling (90-125 min) and increased plasma membrane permeability (150-240 min). After a 30 min exposure to 1 mM hydrogen peroxide, cellular plating efficiency, measured at 24 h, was reduced by 50% (P less than .001). These changes were accelerated, although their order of appearance was unchanged, at higher concentrations of hydrogen peroxide. We conclude that functional and morphologic expressions of cellular injury in this model occur in a defined sequence with plasma membrane depolarization representing the earliest marker of membrane injury during hydrogen peroxide exposure.

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. This reports describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of /sup 35/S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A. Incubation of the purified enzyme with (/sup 3/H)DFP followed by SDS-PAGE and autoradiography revealed a specifically labeled 38-kDa peptide, the substrate binding subunit. Analysis by high-performance liquid chromatography of the 3-kDa products resulting from the cleavage of /sup 35/S-labeled proANF by the purified enzyme revealed, as previously described with whole serum, two radiolabeled peptides which coeluted with the 28 and 24 amino acid C-terminal peptides. Thesemore » observations imply a precursor-product relationship, with the initial cleavage of proANF to the 28 amino acid peptide, which is then cleaved to the 24 amino acid peptide. These studies indicate that the majority of proANF cleavage activity found in rat serum is represented by that of a distinct serine protease whose properties are different from a variety of well-characterized proteases. The role of this protease in the in vivo processing of proANF remains to be defined.« less

A model for oxytocin has been previously proposed in which residues 3 and 4 occupy the corner positions in a beta turn (Urry, D. W., & Walter, R (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 956). The analogue [Pro(3),Gly(4)]-oxytocin (PGO) was used in proton magnetic resonance (1H NMR) studies designed to probe the contribution of these corner positions in forming a beta turn. Comparison of various 1H NMR parameters obtained at 220 MHz for backbone amide protons of PGO with those for the corresponding protons of oxytocin suggests that, to a first approximation, these two peptides may have similar backbone conformations in )CD3)2SO. Theoretically, the L-Pro lead toGly sequence in positions 3 and 4 of PGO would allow the formation of either a type I or type II beta turn. The two coupling constants between vicinal amide and C alpha protons for Gly(4) of PGO in (CD3)2SO are compatible with a type II beta turn in which 04, the dihedral angle about the bond between the backbone C alpha and N' atoms of Gly4, is appromximately +92 degrees, but not with a type I beta turn. A survey of peptides studied in other laboratories by X-ray and/or 1H NMR with reported type II beta turns with L-Pro lead toGly or Gly lead to Gly sequences in the corner positions revealed a close correlation between chemical shifts and vicinal coupling constants for the glycl residue in the second corner position. It is suggested that this criterion can form an additional basis for the characterization of beta turns. More studies are needed to determine the particular type of beta turn manifest in the cyclic moiety of oxytocin per se, although a reasonable working hypothesis is that oxytocin, similar to PGO, has a type II beta turn.

The ability of the neurohypophyseal hormones and related synthetic peptides to potentiate the action of synthetic ovine corticotropin releasing factor (CRF-41) was examined using male rats whose endogenous CRF release was blocked with chlorpromazine, morphine and nembutal. CRF potency was clearly related to the pressor activity of the analogues. However, the threshold dose for eliciting a significant corticosterone response with the neurohypophyseal hormones was greater than with CRF-41. When arginine vasopressin (VAP) was coadministered with CRF-41 at subthreshold doses of both peptides, a significant corticosterone response was observed. When the neurohypophyseal hormone analogues were compared with regard to intrinsic CRF bioactivity, it was observed that an L-basic residue in sequence position 8 is necessary for high intrinsic activity. With one exception, 1-Deamino-(9-D-Ala) arginine vasopressin, the ability to potentiate the effect of CRF-41 was related to the intrinsic CRF potency of the analogues. These results support previous reports of in vitro potentiation of CRF-41 by AVP and point out the complexity of CRF-neurohypophyseal hormone interaction in vivo.

We developed an improved in vivo bioassay for corticotropin releasing factor (CRF) by modifying the injection schedule in the standard chlorpromazine-morphine-pentobarbital assay procedure. A combined injection of chlorpromazine and morphine followed 75 min later by injection of pentobarbital produced low basal levels of corticosterone and rendered the animals highly sensitive to synthetic CRF but insensitive to the stress of ether or histamine. The lowest dose of CRF that significantly elevated plasma corticosterone levels was 0.01 micrograms/kg. Using this assay, we studied CRF-arginine vasopressin (AVP) interactions at doses that were expected to raise systemic peptide concentrations to levels measured in hypophysial portal blood. The threshold for a significant corticosterone response was found to be at least 250-fold lower for CRF-41 than for AVP. The order in which CRF and AVP are injected was also found to be important, potentiation being greater if CRF was given first. In addition, rats deprived of water for 24 hr were more sensitive to CRF than normally hydrated animals.

The effects of three analogs of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) were tested in several behavioral tests after peripheral injection in rats. In the passive avoidance test, rats injected SC with 1 mg/kg Ala-MIF-1 or Phe-MIF-1 entered the second compartment of two-chamber shuttle box significantly faster than did rats receiving diluent. Leu-MIF-1 failed to produce significantly faster entry compared to diluent. None of the peptides significantly affected ambulation, rearing, or defecation. Flinch and escape thresholds were not affected by Ala-MIF-1, the only analog tested for this behavior. The results demonstrate that some analogs of Tyr-MIF-1 can exert behavioral effects similar to those exerted by the parent compound whereas other analogs resemble MIF-1 in being inactive under these experimental conditions.

The diurnal response to ovine corticotropin-releasing factor (CRF-41), arginine vasopressin (AVP), and adrenocorticotropic hormone (ACTH) was studied in rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine sulfate, and pentobarbital sodium. This procedure resulted in a markedly attenuated circadian rhythm at base-line levels of plasma corticosterone and ACTH. Decreased pituitary responsiveness to CRF-41 and AVP at 0400 compared with 1600 was observed. The plasma corticosterone response 30 min after intravenous injection of ovine CRF-41 (0.1 microgram/kg) or AVP (5.0 micrograms/kg) remained nearly constant over the major portion of the 24-h light-dark cycle. However, in the early morning (0400), 2 h before lights on, there was an approximately threefold decrease in response. The time of this decrease in response coincided with the normal decline in the concentrations of plasma corticosterone and ACTH. Rats exposed to constant darkness for 10 days continued to show a significantly greater response to CRF or AVP at 1600 than at 0400. In contrast, rats exposed to constant light for 10 days failed to demonstrate a differential response to CRF or AVP at different times of the day. These results demonstrate that there is a diurnal rhythm in pituitary response to CRF and AVP.

The naturally occurring brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was tested for its ability to block and reverse the actions of morphine in the tail-flick test. Injected peripherally either 10 minutes before or after morphine, Tyr-MIF-1, like MIF-1, was found to significantly reduce the antinociceptive actions of morphine on thermal pain. The results indicate that Tyr-MIF-1 may act, in part, as an endogenous opiate antagonist.

The antiopiate activities of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2 ) and some of its representative analogs were tested in two animal models of antinociception. Doses of the tetrapeptides as low as 0.001 mg/kg injected peripherally could block the analgesic effects of morphine in both the tail-flick test of mild thermal pain induced by heat and the scratching test of mild chemical pain induced by hypertonic saline. These tetrapeptides showed cross-reactivity in the radioimmunoassay ( (RIA) used to identify the presence of Tyr-MIF-1 in brain extracts and in the brain membrane binding assay. Only Tyr-MIF-1, however, eluted at the position of the immunoreactive peak after gel filtration chromotography and high performance liquid chromotography (HPLC). The results support the concept that peptides with anti-opiate activity can exist in the brain.

Hypothalamic CRF-like immunoreactivity was measured in the a.m. and p.m., after systemic dexamethasone administration or after either stress in adult male rats. Measurement of plasma corticosterone levels revealed the expected circadian rhythmicity, suppression after dexamethasone administration and increase after ether stress. The hypothalamic content of CRF-like immunoreactivity was significantly decreased in the p.m. and after dexamethasone administration. However, no change in hypothalamic CRF-like immunoreactivity was observed after ether stress. The results are consistent with an increased release in the p.m. and decreased synthesis of hypothalamic CRF after systemic dexamethasone administration. The observation that there is no change in content of hypothalamic CRF-like immunoreactivity after ether stress could be due to the fact that the animals were stressed by handling. The results show that this immunoreactivity present in the hypothalamus is altered by changes in the hypothalamic-pituitaryadrenal axis and thus suggest that this peptide is a physiologically significant CRF in the rat.

OBJECTIVES Given the importance of inflammation in atherosclerosis, we sought to determine if atherosclerotic plaque inflammation could be measured noninvasively in humans using positron emission tomography (PET). BACKGROUND Earlier PET studies using fluorodeoxyglucose (FDG) demonstrated increased FDG uptake in atherosclerotic plaques. Here we tested the ability of FDG-PET to measure carotid plaque inflammation in patients who subsequently underwent carotid endarterectomy (CEA). METHODS Seventeen patients with severe carotid stenoses underwent FDG-PET imaging 3 h after FDG administration (13 to 25 mCi), after which carotid plaque FDG uptake was determined as the ratio of plaque to blood activity (target to background ratio, TBR). Less than 1 month after imaging, subjects underwent CEA, after which carotid specimens were processed to identify macrophages (staining with anti-CD68 antibodies). RESULTS There was a significant correlation between the PET signal from the carotid plaques and the macrophage staining from the corresponding histologic sections (r = 0.70; p < 0.0001). When mean FDG uptake (mean TBR) was compared with mean inflammation (mean percentage CD68 staining) for each of the 17 patients, the correlation was even stronger (r = 0.85; p < 0.0001). Fluorodeoxyglucose uptake did not correlate with plaque area, plaque thickness, or area of smooth muscle cell staining. CONCLUSIONS We established that FDG-PET imaging can be used to assess the severity of inflammation in carotid plaques in patients. If subsequent natural history studies link increased FDG-PET activity in carotid arteries with clinical events, this noninvasive measure could be used to identify a subset of patients with carotid atherosclerosis in need of intensified medical therapy or carotid artery intervention to prevent stroke.

ObjectivesWe sought to use positron emission tomography (PET) to test the hypothesis that hyperhomocysteinemia adversely effects coronary microvascular dilator function.BackgroundHyperhomocysteinemia is associated with abnormal endothelium-dependent vasodilation in peripheral human arteries. However, its effect on the coronary circulation is not known.MethodsEighteen healthy humans, age 24 to 56 years, were enrolled in a double-blind, crossover trial. Basal and adenosine-stimulated myocardial blood flow (MBF) was determined by PET: after ingestion of placebo and after methionine-induced hyperhomocysteinemia. Further, brachial ultrasonography was used to assess flow-mediated vasodilation. Additionally, to assess the role of nitric oxide (NO) in adenosine-mediated vasodilation, the MBF response to adenosine was measured in the presence and absence of the NO synthase antagonist NG-monomethyl-l-arginine (l-NMMA) (0.3 mg/kg/min intravenously).ResultsHyperhomocysteinemia resulted in a reduction in the MBF dose-response curve to adenosine (p < 0.05). This was most apparent with low dose adenosine, where MBF augmentation was significantly blunted during hyperhomocysteinemia (1.06 ± 1.00 ml/min/g vs. 0.58 ± 0.78 ml/min/g, placebo vs. methionine, p < 0.05). Similarly, flow-mediated brachial artery vasodilation was impaired during hyperhomocysteinemia (4.4 ± 2.6% vs. 2.6 ± 2.3%, placebo vs. methionine, p < 0.05). In a separate series of experiments, MBF during adenosine was reduced in the presence of l-NMMA (p < 0.05 analysis of variance). This was most apparent at the low dose of adenosine, where MBF response to adenosine was blunted in the presence of l-NMMA (2.08 ± 1.34 ml/min/g vs. 1.48 ± 1.32 ml/min/g, placebo vs. l-NMMA, p < 0.05).ConclusionsThe data, therefore, support the hypothesis that acute hyperhomocysteinemia impairs microvascular dilation in the human coronary circulation as a result of reduced NO bioavailability.

CRF-like immunoreactivity was measured by radioimmunoassay in the brains and gastroenteropancreatic tract of normal rabbits. It was detected in the brain, with the highest concentration being found in the ventral hypothalamus. The distribution of immunoreactivity was much more limited in the rabbit brain than in the rat brain, with substantial amounts of peptide detected only in areas of close proximity to the hypothalamus, e.g., thalamus, preoptic area, midbrain and amygdala. In addition, the extrahypothalamic immunoreactivity was slightly retarded on Sephadex G-50 chromatography relative to rat CRF-like immunoreactivity and synthetic ovine CRF. No apparent CRF-like immunoreactivity was detected in boiling water extracts of lung, pancreas, duodenum or antrum. These data in conjunction with a previous report of void volume immunoreactivity on Sephadex G-50 only in the hypothalamus suggest that CRF is synthesized only in the hypothalamus and is not a member of the class of peptides found throughout the gastroenteropancreatic tract and the central nervous system.

Myocarditis may be difficult to diagnose due to the paucity of common clinical and laboratory findings. The gold standard for diagnosis of acute myocarditis is the histopathological examination of endomyocardial biopsy samples for evidence of myocyte necrosis with concurrent cellular infiltrates [1]. Although this method is highly specific, endomyocardial biopsy is relatively insensitive because of the limited number of biopsy samples which can be obtained from each patient, and the focal nature of the disease in some patients. The disease may only cause necrosis in a portion of one ventricle. Since the biopsy can only sample the endocardium, necrosis involving only the epicardium or mid-portion of the myocardium cannot be appreciated from histological examination of the specimen. A method of evaluating necrosis at any location in the myocardium would help to identify patients with myocarditis. To determine whether a radioimmunoscintigraphic approach that is highly specific for delineation and visualization of acute myocardial necrosis in myocardial infarction [2–10] could also serve as a noninvasive method for the diagnosis of acute myocarditis, we studied a series of patients scheduled for ventricular biopsy who were suspected of having acute myocarditis.

High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with 125I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.

Levels of hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI) were measured by radioimmunuoassay (RIA) over a 24 hour light-dark cycle and found to exhibit two peaks. One peak was detected at 1100 hr and a secondary smaller peak was found at 2000 hr. The trough between the two peaks was detected at 1700 hr which coincided with the peak in plasma corticosterone levels. The results are consistent with a decreased level of hypothalamic CRF-LI at 1700 hr reflecting an increased release of peptide followed successively by the release of ACTH and corticosterone.

It has been reported that delta-sleep-inducing peptide (DSIP) can affect several activities other than sleep, including reduction of stress. We studied the effects of this nonapeptide on corticotropin releasing factor (CRF)-stimulated release of corticosterone in rats treated with chlorpromazine-morphine-pentobarbital. Significant reduction of corticosterone levels were observed after intravenous injection of 5-30 micrograms/kg DSIP. No effect of DSIP was found on the corticosterone release elicited by injection of adrenocorticotropic hormone. The results suggest that DSIP attenuates the effects of CRF at the level of the pituitary.

[8-D-Arginine]vasopressin (DAVP), an active synthetic analog of the naturally occurring neurohypophyseal hormone [8-L-arginine]vasopressin (AVP), was studied in D 2 O at pD 3.3 and 23°C by 1 H nuclear magnetic resonance (NMR) spectroscopy, and chemical shifts (δs) and some of the coupling constants for the nonlabile protons (the protons that do not readily exchange for deuterons in the solvent) were determined and compared to those previously reported by us for AVP in D 2 O at pD 3.8 and 20°C ( Wyssbrod et al., 1979a , b ). Values of δ of corresponding protons in DAVP and AVP are quite similar, and small perturbations in these values for protons in the prolyl-7 and glycyl-9 residues in going from AVP to DAVP can be ascribed to the change in chirality (handedness) at the C α of the adjacent arginyl-8 residue. That the observed perturbations are relatively small (⩽ 0.03 ppm) is probably related to extensive conformational interconversion (averaging) in residues 7-9, which comprise the tail moiety, in both peptides. The conformational states of residues 1-6, which comprise the ring moiety, appear to be, for all practical purposes, identical in both peptides.

T WO RADIONUCLIDES, thallium-201 and technetium-99m, have assumed preeminence in cardiovascular nuclear medicine because their photon energies, kinetics, and dosimetry are well-suited to make three major physiologic measurements: perfusion, function, and viability. The photon energies of these nuclides are well matched to the characteristics of the Anger camera, which has optimum efficiency and resolution for energies between 100 and 200 keV. The spatial resolution of the “system” for the average cardiovascular nuclear medicine study (radiopharmaceutical, collimator, camera, and computer) at the depth of the heart is approximately 1 cm full width half maximum (FWHM). Against this background, new radiopharmaceuticals suggested for the evaluation of the heart (Table 1) must offer an improvement in dosimetry and resolution, or provide data on a previously unmeasurable parameter. This review will concentrate on the new radiopharmaceuticals that have met two criteria: (1) experience with the radiopharmaceutical in human subjects, and (2) some likelihood of commercial production of the agent for widespread clinical use. Although equilibrium studies provide high quality data for evaluation of regional and global cardiac function, a high photon flux first pass approach, which could evaluate ventricular function during breathholding (to eliminate respiratory motion), would improve resolution. The ideal first pass radionuclide should have a short physical halflife, have no particulate emissions, produce photons of 100 to 200 keV, and decay to a stable daughter. Such an agent would permit multiple measurements to be performed in each view and would allow repetitive determinations of ventricular function during the action of drugs, exercise, or physiologic maneuvers (Valsalva, etc). The following agents have some of these properties and may supplant Tc-99m as the radionuclide for first pass studies.

One experimental approach to determining the dynamic interconversion around a single bond is to measure multiple vicinal NMR couplings about it. This approach (“circumjacent coupling”) has been extensively applied by us to investigate x1 angles in the side chains of residues in the peptide hormones oxytocin and [8–arginine] vasopressin. The criteria for discriminating rotamer-averaged and predominantly fixed torsion angles have been developed. This approach provides detailed probes of conformation in the peptides, demonstrating the apparent conformational independence of the terminal tripeptide section, the insensitivity of side chain conformation to adjacent substitutions of residues, and the relative rigidity of the disulfide bridge region in the rings of these peptides.

The effect of intraperitoneal administration of cocaine on the concentrations of hypothalamic corticotropin releasing factor like-immunoreactivity (CRF-LI), plasma ACTH, beta-endorphin, and corticosterone was investigated. Groups of rats were injected with 20 mg/kg cocaine HCl or 0.9% NaCl and then killed 0, 10, 20, 30 or 60 minutes later. Hypothalamic CRF-LI, plasma ACTH, beta-endorphin, and corticosterone concentrations were determined by radioimmunoassay. A significant increase in plasma ACTH, beta-endorphin, and corticosterone concentrations was observed after cocaine administration. In contrast, cocaine had no significant effect on hypothalamic CRF-LI concentrations. Intravenous administration of 0.5 and 2.0 mg/kg cocaine to rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine, and pentobarbital elicited a significant increase in plasma corticosterone concentrations. These results demonstrate that cocaine induces the release of ACTH, beta-endorphin, and corticosterone and suggest that this response is mediated at the pituitary level.

Delta sleep-inducing peptide-like immunoreactivity (DSIP-LI) was measured by radioimmunoassay (RIA) in invidual brain areas of rats running a 12-choice maze. One of the areas found to contain a high level of DSIP-LI was the thalamus, where a distinct peak of immunoreactivity eluted at the position of the synthetic DSIP noapeptide after high performance liquid chromatography (HPLC). On each of the two days of testing, DSIP-LI in the thalamus correlated significantly with running times in the maze. The results suggest an association of behavior with the levels of peptide in a region of the brain.

[7-(3, 4-Dehydroproline), 8-arginine]vasopressin ([Δ3-Pro7]AVP), an active analog of the naturally occurring neurohypophyseal hormone [8-arginine]vasopressin (AVP), was studied in D2O at pD 3.3 and 23° by 1H n.m.r. spectroscopy, and chemical shifts (SdLs) and some of the coupling constants for the nonlabile protons (1Hs) were determined and compared to those previously reported by us for AVP in D2O at pD 3.8 and 20° (Wyssbrod et al., 7979a, b). Double- resonance difference spectroscopy (Gibbons et al., 1975) was used to reveal positions of certain resonances obscured by overlap with resonances of other 1Hs. Values of δ of corresponding protons in [Δ3-Pro7]AVP and AVP are quite similar, and the small differences that are observed can be explained on the basis of (a) magnetic anisotropy from the double bond in the Δ3 -prolyl residue, (b) a slight difference in acidity of the samples in the two studies, or (c) uncertainty in the experimentally determined value of δ. Values of δ for the 1Hs in the Δ3-prolyl and prolyl residues in the two peptides are reported. Our tentative conclusion is that the differences in reported biological activities of [Δ3-Pro7]AVP and AVP (Botos et al., 1979) are probably not [Δ3-PRO7, ARG8] VASOPRESSIN related to a perturbation in the conformational state of the backbone, but that they might be explained, in part, by a change in the conformation of the five- membered ring of residue 7 and, in part, by π-π interaction between the 3-pyrroline ring of the Δ3-prolyl residue of [Δ3-Pro7]AVP and the receptor involved in the antidiuretic response but not the receptor involved in the pressor response, in accordance with the suggestion of Walter (1977) that π-π interactions might, in some cases, be involved in binding of a peptide to its receptor.

Evidence is presented that peptides may occur in aggregated form. Addition of 125I-Tyr-DSIP to serum resulted in four peaks after gel filtration chromatography on a column of Sephadex G-25. One of the peaks (C) eluted at the same position as the labeled peptide standard. Two Peaks (A and B) eluted before the standard and one (peak D) afterwards. The first peak (A) eluted at void volume, a position expected for labeled peptide bound to protein. The other two peaks (B and D), corresponding to smaller molecular size material, were greatly reduced by addition of glacial acetic acid or the chelating agent 1,10-phenanthroline before or even after mixing of the peptide with serum. Iron was one of the ions found to interact with 125I-Tyr-DSIP, and chromatography of a mixture of ferric chloride and peptide without serum resulted in the additional formation of peak B. A substantial portion of peaks A, B, and C (but not D) reacted with a specific DSIP antibody, indicating the presence of intact peptide. The results are consistent with the concept that peptides may occur in multiple forms.

The effect of constant light and constant dark on the circadian rhythm of the concentrations of hypothalamic corticotropin-releasing-factor-like immunoreactivity (CRF-LI), plasma ACTH, and corticosterone was investigated. Groups of rats were maintained under normal light-dark, constant light, or constant dark conditions for 10 days. Rats were then killed over a 24-hour time period and hypothalamic CRF-LI, plasma ACTH, and corticosterone concentrations were determined by radioimmunoassay. Under normal light-dark conditions, troughs in hypothalamic CRF-LI concentrations coincided with peaks in plasma ACTH and corticosterone concentrations. In rats housed under constant dark conditions for 10 days, higher hypothalamic CRF-LI concentrations were detected at 20.00, 24.00 and 04.00 h than at 08.00, 12.00 and 16.00 h. These relatively high hypothalamic CRF-LI concentrations coincided with relatively low plasma ACTH concentrations. The amplitude of plasma ACTH concentrations was markedly attenuated compared to levels of rats housed under normal light-dark conditions. The rats exposed to constant dark continued to demonstrate higher plasma corticosterone concentrations post meridiem than ante meridiem. The peak in plasma corticosterone coincided with the peak in plasma ACTH concentrations; however, the amplitude was normal. In rats maintained in constant light for 10 days, a decrease in hypothalamic CRF-LI concentrations at 20.00 h coincided with a peak in plasma ACTH. The peak in plasma ACTH concentrations was not associated with a peak in plasma corticosterone concentrations. The rhythm of plasma corticosterone concentrations was dramatically attenuated and phase-shifted. Together, these findings indicate that alterations of normal light-dark conditions result in changes in the circadian variation in hypothalamic CRF-LI, plasma ACTH, and corticosterone concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Although delta-sleep inducing peptide was isolated and characterized several years ago, no definitive evidence has been presented for the natural existence of the free peptide. Several attempts at the partial characterization of DSIP-like immunoreactivity (DSIP-L1) have indicated that a small part of the total immunoreactivity is probably present as the free nonapeptide. Using gel chromatography (Sephadex G-100) and subsequent high performance liquid chromatography on rabbit, human, rat and dog plasma, we now show a distinct peak of DS IP-LI that has the same elution position as synthetic DSIP. Free DSIP was also found in human CSF, whereas in human urine most of the small molecular weight DSIP-LI eluted at a position corresponding to DSIP-P, the phosphorylated analog of DSIP. A newly developed antibody recognizing primarily small molecular weight DSIP-LI was used in a modified, rapid assay to facilitate demonstration of the natural occurrence of free DSIP.

Hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI), plasma ACTH and corticosterone levels were measured by radioimmunoassay over a two hour period of restraint stress. The results of this study demonstrate a significant decrease in hypothalamic CRF-LI levels 15 and 30 minutes after the start of restraint stress which is followed by a significant increase at 60 minutes that is abolished by cycloheximide pretreatment. Plasma ACTH and corticosterone levels were significantly elevated after 15, 30, 60, 90, and 120 minutes of restraint stress. These results are consistent with a release of CRF from the hypothalamus during stress. The cycloheximide-sensitive increase in hypothalamic CRF-LI indicates that synthesis of CRF-41 occurs during prolonged stress. These results suggest that the response of an organism to exposure to a long-term, high intensity stress involves both the release and synthesis of CRF-41.

Cell membrane potential was measured with a flow cytometer by quantitating the intracellular accumulation of a fluorescent cationic carbocyanine dye. We used this system to demonstrate depolarization upon the addition of hydrogen peroxide (10-1,000 microM) and ferrous chloride (25-100 microM) to cultures of either neonatal rat myocardial or LLC-PK1 renal epithelial cells. Ferrous chloride-induced depolarization was prevented by superoxide dismutase, catalase and dimethyl sulfoxide, suggesting roles for the superoxide anion, hydrogen peroxide and the hydroxyl radical in effecting this depolarization, possibly through a Fenton-type reaction mechanism. Supplementation of either cell type with 2 microM tocopherol acid succinate during growth in tissue culture, prior to exposure to the oxidizing agent, decreased the magnitude of the depolarization in both cell types. The results are consistent with a role for tocopherols in scavenging free radical species responsible for the depolarization of the cell membrane.

Corticotropin releasing factor (CRF) was recently isolated from ovine hypothalami by its ability to stimulate adrenocorticotropin (ACTH) and β-endorphin release from dispersed rat pituitary cells. In order to study the physiology of this peptide, we have developed a sensitive and specific radioimmunoassay (RIA) for CRF. Synthetic CRF was conjugated to bovine thyroglobulin and emulsified with complete Freund's adjuvant. A suitable antiserum was obtained which showed no crossreactivity with eight naturally occurring peptides. N-Tyr-CRF was iodinated and used as tracer. With this assay, CRF-like immunoreactivity which coeluted with ovine CRF on Sephadex G50 was detected in rat hypothalami.

Tomographic gated blood pool imaging is a natural extension of the technologies of planar gated blood pool scanning and rotating Anger camera single photon emission computed tomography (SPECT). The high photon flux, optimum 140 keV energy, and volume sampling of tomography permit reconstruction of the data in any perspective. The true three-dimensional nature of this process allows the evaluation of regional wall motion of all the cardiac chambers, unencumbered by overlapping structures. The heart can be viewed from any angle, including a long axis, short axis, apical four chamber, and a true inferior view. In addition to evaluation of regional wall motion, precise determination of chamber volumes and ejection fractions is possible. Early clinical experience has demonstrated the superiority of tomographic gated blood pool imaging over planar blood pool imaging for precisely defining subtle functional abnormalities. The enormous amount of data generated by this procedure taxes the capacity of most nuclear medicine computer systems. However, the availability of 32-bit processors and large amounts of image memory in new machines should ultimately reduce this processing time to less than ten minutes. The combination of complete visualization and quantitation suggests that a renaissance for blood pool imaging is on the horizon.