High-resolution computed tomography of the chest in children with cystic fibrosis: support for use as an outcome surrogate.

BACKGROUND: Outcome surrogates are indicators that reflect, rather than directly measure, patient benefit. In order to provide useful results, however, outcome surrogates must be carefully chosen and must meet specific criteria. OBJECTIVE: To support development of high-resolution computed tomography (HRCT) as an outcome surrogate in cystic fibrosis (CF) by demonstrating the ability of HRCT to show short-term improvement in the appearance of the lungs in children with CF. MATERIALS AND METHODS: HRCT was performed at admission and after discharge on 8 children during 15 admissions for acute pulmonary exacerbation of CF. Three radiologists scored each study separately, then compared admission and discharge pairs. RESULTS: HRCT scores improved in 13/15 admissions. Mean score decreased from 25 to 22. The decrease was significant (P = 0.014). Comparison of admission and discharge scans showed improvement in peribronchial thickening (P = 0.007), mucous plugging (P = 0.002), and overall appearance (P = 0.025). CONCLUSION: HRCT has the potential to be a useful outcome surrogate in CF. A necessary attribute of an outcome surrogate is that it improves rapidly with effective therapy. Despite widespread belief among radiologists and pulmonologists that HRCT meets this criterion, no previous report has demonstrated this ability in children. These findings support further development of HRCT as an outcome surrogate in children with CF.

Neuropeptide degradation produces functional inactivation in the crustacean nervous system.

The pentapeptide proctolin (Proct.; Arg-Tyr-Leu-Pro-Thr) is a modulatory transmitter found throughout the crustacean nervous system. No information is available in this system, however, as to how the actions of this peptide are terminated. To study this issue in the crab Cancer borealis, we incubated exogenous proctolin (10(-5) M) with either the thoracic ganglion (TG) or with conditioned saline (CS) that had been preincubated with the TG. We removed aliquots at standard time points for analysis by reverse-phase high-performance liquid chromatography (HPLC). We found that over time the proctolin peak became progressively smaller, while three novel peaks appeared and increased in size. Comigration experiments using HPLC indicated that the major novel peak was Proct. (Tyr-Leu-Pro-Thr), while one of the two minor peaks was Proct. (Leu-Pro-Thr). The other minor peak appeared to be Proct. (Arg-Tyr), based on similar HPLC retention time to synthetic Proct. The reduction in the proctolin peak and the increase in the Proct. peak was prevented by co-incubation of proctolin with any one of several aminopeptidase inhibitors (10(-4) M). Proct. and Proct. appeared to result from a diaminopeptidase-mediated cleavage of proctolin. We tested whether N-terminal cleavage functionally inactivated proctolin by coapplying proctolin (10(-8) M) and individual aminopeptidase inhibitors (10(-5) M) to the isolated stomatogastric ganglion (STG). We found that these inhibitors significantly enhanced the proctolin excitation of the pyloric rhythm. Furthermore, application of synthetic Proct. to the STG had no effect unless high concentrations (> 10(-6) M) were used, and neither Proct. nor Proct. (10(-4) M) influenced the pyloric rhythm. Our results indicate that proctolin is enzymatically degraded and thereby biologically inactivated in the crab nervous system, primarily by extracellularly located aminopeptidase activity.

Aplysia peptide neurotransmitters beta-bag cell peptide, Phe-Met-Arg-Phe-amide, and small cardioexcitatory peptide B are rapidly degraded by a leucine aminopeptidase-like activity in hemolymph.

We have been investigating the role of proteolytic enzymes in the inactivation of peptide neurotransmitters in the marine snail Aplysia. Previous studies (Squire, C. R., Talebian, M., Menon, J. G., Dekruyff, S. D., Lee, T. D., Shively, J. E., and Rothman, B. S. (1991) J. Biol. Chem. 266, 22355-22363) showed that neuroactive fragments of the neurotransmitter alpha-bag cell peptide (alpha-BCP) were rapidly degraded (t1/2 = 0.5-2.7 min) in plasma, hemolymph that had been cleared by centrifugation. Degradation was caused by one or more enzymes resembling mammalian leucine amino-peptidase (LAP, EC 3.4.11.1). In this report we show that three other Aplysia peptide neurotransmitters, beta-BCP(1-5) (Arg-Leu-Arg-Phe-His), FMRFa (Phe-Met-Arg-Phe-amide), and SCPB(1-9) (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide) are rapidly degraded (t1/2 = 0.3-2.4 min) in plasma by apparently the same LAP-like enzyme(s). Our findings strongly suggest that the LAP-like enzyme(s), by means of its broad substrate specificity and access to the extracellular spaces of the nervous system in vivo, plays a significant role in the inactivation of many Aplysia peptide neurotransmitters, and they raise the possibility that proteolytic enzymes in the extracellular fluid contribute significantly to the inactivation of peptide neurotransmitters in other animal species.

Structure-activity relationship of the neurotransmitter alpha-bag cell peptide on Aplysia LUQ neurons: implications regarding its inactivation in the extracellular space.

Alpha-bag cell peptide [alpha-BCP (Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu)] is a neurotransmitter that mediates bag cell-induced inhibition of left-upper-quadrant (LUQ) neurons L2, L3, L4, and L6 in the abdominal ganglion of Aplysia. Our recent biochemical studies have shown that alpha-BCP[1-9] is cleaved into alpha-BCP[1-2], [3-9], [1-5], [6-9], and [7-9] by a combination of three distinct peptidase activities located within the extracellular spaces of the CNS: A diaminopeptidase-IV (DAP-IV)-like enzyme cleaves alpha-BCP[1-9] at the 2-3 peptide bond; a neutral metalloendopeptidase (NEP)-like enzyme cleaves either alpha-BCP[1-9] or alpha-BCP[3-9] at the 5-6 bond; an aminopeptidase M-II (APM-II)-like enzyme cleaves alpha-BCP[6-9] at the 6-7 bond, but cleaves neither alpha-BCP[1-9], nor the other ganglionic peptidase products. To further understand the manner in which alpha-BCP is inactivated after release, that is loses its electrophysiological activity, we studied its structure-activity relationship by recording intracellularly from LUQ neurons in isolated abdominal ganglia that were arterially perfused with peptides dissolved in artificial sea water. The effects of alpha-BCP[1-9] and 15 of its fragments ([1-8], [1-7], [1-6], [1-5], [2-9], [3-9], [3-8], [6-9], [7-9], [8-9], [6-7], [6-8], [1-2], Phe, Tyr) indicated that the sequence Phe6-Tyr7 was both necessary and sufficient to produce LUQ inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Leucine aminopeptidase-like activity in Aplysia hemolymph rapidly degrades biologically active alpha-bag cell peptide fragments.

We have investigated the role that proteolytic enzymes in Aplysia hemolymph play in the inactivation of the neurotransmitter alpha-bag cell peptide (alpha-BCP(1-9), Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu). alpha-BCP fragments containing Pro in positions 1 or 2, or Tyr in position 1, were degraded relatively slowly (half-life, t1/2 = 10-64 min), whereas fragments lacking these residues were degraded relatively rapidly (t1/2 = 0.5-2.7 min). Of 12 peptidase inhibitors tested, only bestatin, amastatin, and phenanthroline significantly inhibited alpha-BCP(3-9) degradation. alpha-BCP(3-9) yielded only four observable cleavage products (in order of decreasing abundance at early time points): alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), and alpha-BCP(7-9). Degradation of alpha-BCP(3-9), alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), or alpha-BCP(7-9) was strongly inhibited by bestatin, moderately inhibited by amastatin, and not inhibited by arphramenine B. The rates of degradation of eight alpha-BCP fragments and three other peptides in plasma were well correlated with their rates of degradation in mammalian leucine aminopeptidase (LAP, EC 3.4.11.1). Collectively our data support the following ideas. 1) In hemolymph one or more LAP-like enzymes rapidly and sequentially cleave alpha-BCP(3-9) or other small peptides lacking Pro at positions 1 or 2 or Tyr at position 1. 2) LAP-like peptidases in hemolymph may act in concert with previously described ganglionic peptidases to degrade neurally released alpha-BCP(1-9) and alpha-BCP(1-8) into inactive fragments.

Separation and determination of diesel contaminants in various fish products by capillary gas chromatography.

A semiquantitative capillary column gas chromatographic method is described for the determination of diesel fuel contamination in various canned seafood products. The diesel contaminants are separated from the fish sample by steam distillation, with little carry-over of interfering intrinsic materials such as fish oils. The diesel fuel is extracted from the condensate with n-hexane, and the extract is analyzed on an SPB-1 fused silica capillary column. The efficiency of recovery of diesel fuel added to canned seafood at levels of 40-400 ppt ranged from 72 to 102%. With the additional step of concentrating the hexane extract, the sensitivity of this procedure may be increased at least 10-fold. This procedure can detect the differences among diesel fuel grades No. 1, 2, and 5, and variations within diesel grade No. 2, and thus may be useful in determining the type of petroleum contaminants present in various canned fish products.

Presence of immunoreactive alpha-bag cell peptide[1-8] in bag cell neurons of Aplysia suggests novel carboxypeptidase processing of neuropeptides.

alpha-bag cell peptide (alpha BCP) is a putative neurotransmitter released from bag cell neurons of the marine mollusc Aplysia. alpha BCP is present in bag cell extracts and releasate from bag cells in two neuroactive forms: alpha BCP[1-9] and alpha BCP[1-8]. alpha BCP[1-8] is 30 times as potent as [1-9] in inhibiting target neurons, suggesting that both forms of the peptide serve as neurotransmitters. However, biochemical and molecular genetic data suggest that only alpha BCP[1-9] is originally cleaved directly from a larger precursor protein and that generation of alpha BCP[1-8] would require an unusual C-terminal leucine cleavage of alpha BCP[1-9]. To further ascertain which forms of alpha BCP are normally present in bag cells, we generated highly specific antisera to each peptide. We found intense immunostaining for both peptides in bag cell somata and nerve terminals. Moreover, both forms were stable in bag cell extract for at least 1 hr, which suggests that proteolysis in the extracts had been effectively inhibited. These results suggest that both alpha BCP[1-8] and [1-9] are normally present in bag cell somata and terminals and that a small amount of alpha BCP[1-9] is processed to alpha BCP[1-8] in vesicles before release. The results support the interpretation that the activity of an intravesicular carboxypeptidase generates alpha BCP[1-8] and thereby regulates the amount of inhibitory activity released during a bag cell discharge.

Coexistence of egg-laying hormone and alpha-bag cell peptide in bag cell neurons of Aplysia indicates that they are a peptidergic multitransmitter system.

Double-label immunocytochemistry reveals that immunoreactivity for two putative peptide transmitters, egg-laying hormone and alpha-bag cell peptide, co-exist in most bag cell somata and processes in the abdominal ganglion of the marine mollusc Aplysia. Together with previous physiological and biochemical data these findings indicate that the neuroendocrine bag cells are a multitransmitter system which utilizes two or more peptides derived from a common precursor.

The bag cells of Aplysia as a multitransmitter system: identification of alpha bag cell peptide as a second neurotransmitter.

The bag cell neurons of the marine mollusk, Aplysia, are a putative multitransmitter system that utilizes two or more peptide transmitters derived from a common precursor protein. Two putative transmitters are egg-laying hormone (ELH), a 36 amino acid peptide that induces egg laying and mediates bag cell-induced excitatory effects on certain abdominal ganglion neurons, and alpha-bag cell peptide (alpha BCP), which mimics bag cell-induced inhibition of the left upper quadrant (LUQ) neurons and the depolarization of the bag cells that occurs during the bag cell burst discharge. Alpha BCP was previously purified from bag cell extracts in three neuroactive forms: alpha BCP(1-9), a nine amino acid peptide encoded on the ELH/BCP precursor protein, and two NH2-terminal fragments, alpha BCP(1-8) and alpha BCP(1-7). Analyzing bag cell-induced inhibition of LUQ neurons, we report here that alpha BCP fulfills the main criteria for transmitter identification: stimulation of individual bag cells produces inhibition of the neurons; inhibitory activity is present in releasate collected following an elicited bag cell burst discharge in the presence of protease inhibitors; alpha BCP(1-9) and alpha BCP(1-8) are detected in the releasate in the presence of protease inhibitors; alpha BCP is rapidly inactivated after release, as indicated by the lack of detectable alpha BCP or inhibitory activity in the releasate in the absence of protease inhibitors, and by the increase in potency of the arterially perfused peptide in the presence of protease inhibitors; alpha BCP and the endogenously released transmitter produce apparently identical changes in membrane conductance; bag cell-induced inhibition is reduced or abolished following desensitization of the inhibitory response by long-term application of high concentrations of alpha BCP. The results provide additional evidence that the bag cells are a multitransmitter system and also suggest that many of the physiological properties of alpha BCP-mediated neurotransmission differ from those of ELH. First, unlike ELH, alpha BCP is rapidly inactivated after release. Second, alpha BCP(1-9) may be activated by carboxypeptidase cleavage since alpha BCP(1-8) and alpha BCP(1-7) are 30 and 10X as potent, respectively, as alpha BCP(1-9). Third, the inhibitory action of alpha BCP on its targets has a more rapid onset and a shorter time course than the excitatory actions of ELH. Thus, alpha BCP may diffuse to less distant targets than ELH and serve to regulate the more rapidly occurring neural events underlying egg-laying behavior.

Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica.

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.

Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R3-R14 of Aplysia.

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R3-R14, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R3-R11 have been postulated to play a role in cardiovascular control, peptide II was without effect at less than or equal to 10(-4) M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.

Routine fast atom bombardment mass spectral analysis of high molecular weight peptides--atrial gland peptides from Aplysia californica.

Three peptides isolated from the atrial glands of Aplysia californica were analysed by Fast Atom Bombardment Mass Spectrometry. Survey scans over the mass range 1650 to 7500 at 500 resolution were used to locate signals for the protonated molecular ion and two subunits which result from cleavage of a single disulfide bond. A more accurate mass determination was made by accumulating scans over a narrow mass range. The amounts of sample used for each measurement ranged between 10 and 30 pmoles. Measured mass values are within 0.5 amu of calculated average molecular weights. Results illustrate the utility of the technique for accurate molecular weight determinations on limited quantities of high molecular weight peptides.

Nonsynaptic characteristics of neurotransmission mediated by egg-laying hormone in the abdominal ganglion of Aplysia.

The bag cell neurons of the marine mollusk Aplysia are a putative multitransmitter system which utilizes two or more neuropeptides that are enzymatically cleaved from a common precursor protein. It has been proposed that one of the neuropeptides, egg-laying hormone (ELH), acts nonsynaptically as a neurotransmitter in the abdominal ganglion by diffusing long distances to target neurons compared to conventional transmitters acting at synapses. To test this idea further, we investigated the physiological properties of neurotransmission mediated by ELH. We found that ELH acts directly to duplicate two types of responses produced by a burst discharge of the bag cells: prolonged excitation of LB and LC cells, and the previously described effect of ELH, burst augmentation of cell R15. Analysis of perfusate collected after electrical stimulation of the bag cells showed that the peptide is released in sufficient quantity to diffuse long distances within the ganglion without being completely inactivated. To mimic the way the peptide is thought to be released physiologically, ELH was arterially perfused into the ganglion. The response normally produced by bag cell activity was duplicated by 0.5 to 1.0 microM concentrations of ELH and showed no rapid desensitization. ELH had no effect on cells that are unaffected by bag cell activity and no effect on cells that are inhibited (LUQ cells) or transiently excited (cells L1 and R1) by bag cell activity. Acidic peptide, another peptide encoded on the ELH precursor protein, was found to be synthesized and released by the bag cells, but it had no effect on the cells we tested. We conclude that the combined properties of ELH neurotransmission resemble the properties of transmission at autonomic nerve endings on cardiac and smooth muscle rather than those of conventional synaptic transmission. ELH released from bag cells is dispersed throughout the interstitial and vascular spaces of the ganglion to produce responses in the cells that have receptors for the peptide. The results also suggest that ELH mediates only a subset of the responses induced by bag cell activity; they are consistent with data indicating that the other responses are mediated by other bag cell peptides derived from the same precursor protein as ELH.

Egg-laying hormone: direct action on the ovotestis of Aplysia.

The bag cells are a group of neuroendocrine cells located in the abdominal ganglion of Aplysia californica. These cells induce egg laying in the animal through the release of neurohormone(s). Previous experiments established that an extract of bag cells releases eggs from isolated ovotestis fragments in a dose-dependent manner. (F. E. Dudek and S. S. Tobe, 1978, Gen. Comp. Endocrinol. 36, 618-627.) Experiments presented here were conducted to purify and identify the component(s) of bag cell extract with the egg-releasing activity. Bag cell extracts were fractionated by means of gel-filtration chromatography followed by cation-exchange chromatography. Column eluates were assayed for egg-release activity on isolated ovotestis fragments by the method of Dudek and Tobe (1978). Only one component purified from the crude extract had egg-releasing activity. This component was identified as egg-laying hormone (ELH) based on its purification characteristics, effects on neuronal activity, and migration on thin-layer chromatography. In dose-response studies egg release increased with the concentration of ELH and had a threshold of 8 X 10(-10) M or less. ELH had the same dose-response relationship in egg release assays when present in purified form or as a component of bag cell extract. These data show that ELH acts directly on the ovotestis and that ELH is the only component of bag cell extract with egg-release activity. Taken with the results of other studies (W. D. Branton, S. Arch, T. Smock, and E. Mayeri, 1978. Proc. Nat. Acad. Sci. USA 75, 5732-5736. B. S. Rothman, P. Brownell, and E. Mayeri, 1979. Soc. Neurosci. Abstr. 5, 260. E. Mayeri and B. S. Rothman, 1982.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure and neuronal effects of alpha-bag cell peptide, a second candidate neurotransmitter encoded by a single gene in bag cell neurons of Aplysia.

A discharge of impulse activity in a group of neuroendocrine cells, the bag cells, produces several types of prolonged responses in various identified neurons of the abdominal ganglion of Aplysia. Two excitatory responses are almost certainly mediated by egg-laying hormone, but this peptide cannot account for other responses, such as inhibition of left upper quadrant neurons. We report here the isolation from bag cell clusters of three structurally similar peptides, seven, eight, and nine residues long, that are candidate transmitters for mediating bag cell-induced inhibition. They may also serve as autoexcitatory transmitters since the seven-residue peptide produces a slow depolarization of the bag cells similar to that which occurs during bag cell discharge. The amino acid sequence of the largest peptide, termed alpha-bag cell peptide[1-9], is H-Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu-OH. The other two peptides are identical to alpha-BCP[1-9] except that they lack the COOH-terminal Ser-Leu or leucine residues. The three peptides inhibit left upper quadrant neurons at relative potencies of 10:30:1 (seven-, eight-, and nine-residue peptides, respectively). Recent molecular genetic analysis shows that both alpha-BCP[1-9] and egg-laying hormone are encoded by the same bag cell-specific gene. The multiple neuronal effects of bag cells are therefore likely to be mediated by at least two transmitters that are cleaved from a common precursor molecule.

A single gene encodes multiple neuropeptides mediating a stereotyped behavior.

Egg laying in Aplysia is characterized by a stereotyped behavioral array which is mediated by several neuroactive peptides. We have sequenced two genes encoding the A and B peptides thought to initiate the egg-laying process, as well as a gene encoding egg-laying hormone (ELH) which directly mediates the behavioral array. The three genes share 90% sequence homology and are representatives of a small multigene family. Each gene encodes a protein precursor in which the active peptides are flanked by internal cleavage sites providing the potential to generate multiple small peptides. Each of the three genes consists of sequences homologous to A or B peptide as well as ELH. Although these genes share significant nucleotide homology, they have diverged such that different member genes express functionally related but nonoverlapping sets of neuroactive peptides in different tissues.

Swimming in Aplysia brasiliana: analysis of behavior and neuronal pathways.

Swimming of Aplysia brasiliana was analyzed using time-lapse video and computer graphics techniques to quantify the cyclical movements of different regions of the parapodia. Both the speed of swimming and the period of oscillation are temperature dependent, whereas the metachronal offset is not. The parapodial arterial supply is described; ligation of parapodial arteries does not affect the parapodial motions. Peripheral and central lesions indicated that (1) the anterior parapodial nerve plays the major role in parapodial flapping, (2) a separate neuronal oscillator resides in each pedal ganglion, (3) bilateral coordination is mediated via the pedal commissure, and (4) the swimming "command" pathway is the cerebro-pedal connective. During regular parapodial flapping the speed of level midwater swimming is constant throughout the cycle, suggesting that swimming is not produced by jet propulsion. An alternative propulsion model is advanced.

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies.

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0-7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.