Effects of the copper oxide nanoparticles (CuO NPs) on Galleria mellonella hemocytes.

In this study, 38 nm-sized and flake-like-shaped CuO NPs (10, 50, 100, 150 µg/10 µl/larva) were force-fed to fourth instar (100 ± 20 mg) Galleria mellonella (Lepidoptera: Pyralidae) larvae under the laboratory conditions. The effects of CuO NPs on total hemocyte counts (THCs) and the frequency of viable, mitotic, apoptotic, necrotic, and micronucleated hemocyte indices were detected with the double-staining protocol by hematoxylin and eosin (H&E) stains. The total hemocyte counts (THCs) did not change significantly in G. mellonella larvae at all concentrations for 24 h and 72 h post-force-feeding treatment. The ratio of viable hemocytes decreased at 50, 100, 150 µg/10 µl concentrations in 24 h and 72 h when compared with untreated larvae. The increases in the percentage of mitotic and micronucleated hemocytes were statistically significant at 150 µg/10 µl in 24 h. The results showed that high concentrations (>10 µg/10 µl) of CuO NPs increased the percentage of apoptotic hemocytes in 24 h. 100 and 150 µg/10 µl of CuO NPs caused a significant increase in the percentage of necrotic hemocytes in 24 h. The decrease in the percentage of mitotic hemocytes at 10, 100 and 150 µg/10 µl in 72 h was statistically significant. Apoptotic hemocytes increased and were found to be higher at 100 and 150 µg/10 µl of CuO NPs in 72 h in comparison with the untreated larvae. Finally, we observed an increase in the percentage of necrotic hemocytes at 150 µg/10 µl in 72 h.

Differential gene expression in glioblastoma defined by ADC histogram analysis: relationship to extracellular matrix molecules and survival.

BACKGROUND AND PURPOSE: ADC histogram analysis can stratify outcomes in patients with GBM treated with bevacizumab. Therefore, we compared gene expression between high-versus-low ADC tumors to identify gene expression modules that could underlie this difference and impact patient prognosis. MATERIALS AND METHODS: Up-front bevacizumab-treated patients (N = 38) with newly diagnosed glioblastoma were analyzed by using an ADC histogram approach based on enhancing tumor. Using microarrays, we compared gene expression in high-versus-low ADC tumors in patients subsequently treated with bevacizumab. Tissue sections from a subset of tumors were stained for collagen and collagen-binding proteins. Progression-free and overall survival was determined by using Cox proportional hazard ratios and the Kaplan-Meier method with the log rank test. RESULTS: A total of 13 genes were expressed at 2-fold or greater levels in high- compared with low-ADC tumors at the P < .05 level. Of these, 6 encode for collagen or collagen-binding proteins. High gene expression for the collagen-binding protein decorin was associated with shorter survival (HR, 2.5; P = .03). The pattern and degree of collagen staining were highly variable in both high- and low-ADC tumors. CONCLUSIONS: High-ADC GBMs show greater levels of ECM protein gene expression compared with low-ADC GBMs. It is unclear whether this translates to the accumulation of higher levels of the encoded proteins. However, because ECM molecules could contribute to a proinvasive phenotype, this relationship merits further investigation.

Transforming growth factor beta1 alters synapsin distribution and modulates synaptic depression in Aplysia.

Transforming growth factor beta1 (TGF-beta1) induces long-term synaptic facilitation and long-term increases in excitability in Aplysia. Here we report that this growth factor has acute effects as well. Treatment of pleural-pedal ganglia with TGF-beta1 for 5 min activated mitogen-activated protein kinase (MAPK) and stimulated the phosphorylation of synapsin in a MAPK-dependent manner. This phosphorylation appeared to modulate synapsin distribution in cultured sensory neurons. Control neurons exhibited a punctate distribution of synapsin along neurites, which appeared to represent high concentration aggregates of synapsin. TGF-beta1-treated sensory neurons showed a significant reduction in the number of these puncta, an effect that was blocked by the MAP/ERK kinase inhibitor U0126. The functional consequence of TGF-beta1 was tested by examining its effects on synaptic transmission at the sensorimotor synapse. Application of TGF-beta1 reduced the magnitude of synaptic depression. This effect was dependent on MAPK, consistent with the hypothesis that TGF-1 mobilizes synaptic vesicles through the phosphorylation of synapsin.

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia.

An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V(max) of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation.

Localization of glutamate and glutamate transporters in the sensory neurons of Aplysia.

The sensorimotor synapse of Aplysia has been used extensively to study the cellular and molecular basis for learning and memory. Recent physiologic studies suggest that glutamate may be the excitatory neurotransmitter used by the sensory neurons (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779). We further investigated the hypothesis that glutamate is the excitatory neurotransmitter at this synapse. The somata of sensory neurons in the pleural ganglia showed strong glutamate immunoreactivity. Very intense glutamate immunoreactivity was present in fibers within the neuropil and pleural-pedal connective. Localization of amino acids metabolically related to glutamate was also investigated. Moderate aspartate and glutamine immunoreactivity was present in somata of sensory neurons, but only weak labeling for aspartate and glutamine was present in the neuropil or pleural-pedal connective. In cultured sensory neurons, glutamate immunoreactivity was strong in the somata and processes and was very intense in varicosities; consistent with localization of glutamate in sensory neurons in the intact pleural-pedal ganglion. Cultured sensory neurons showed only weak labeling for aspartate and glutamine. Little or no gamma-aminobutyric acid or glycine immunoreactivity was observed in the pleural-pedal ganglia or in cultured sensory neurons. To further test the hypothesis that the sensory neurons use glutamate as a transmitter, in situ hybridization was performed by using a partial cDNA clone of a putative Aplysia high-affinity glutamate transporter. The sensory neurons, as well as a subset of glia, expressed this mRNA. Known glutamatergic motor neurons B3 and B6 of the buccal ganglion also appeared to express this mRNA. These results, in addition to previous physiological studies (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Trudeau and Castellucci [1993] J Neurophysiol. 70:1221-1230; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779)) establish glutamate as an excitatory neurotransmitter of the sensorimotor synapse.

TGF-beta1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TbetaR-II-like immunoreactivity.

Exogenous recombinant human transforming growth factor beta-1 (TGF-beta1) induced long-term facilitation of Aplysia sensory-motor synapses. In addition, 5-HT-induced facilitation was blocked by application of a soluble fragment of the extracellular portion of the TGF-beta1 type II receptor (TbetaR-II), which presumably acted by scavenging an endogenous TGF-beta1-like molecule. Because TbetaR-II is essential for transmembrane signaling by TGF-beta, we sought to determine whether Aplysia tissues contained TbetaR-II and specifically, whether neurons expressed the receptor. Western blot analysis of Aplysia tissue extracts demonstrated the presence of a TbetaR-II-immunoreactive protein in several tissue types. The expression and distribution of TbetaR-II-immunoreactive proteins in the central nervous system was examined by immunohistochemistry to elucidate sites that may be responsive to TGF-beta1 and thus may play a role in synaptic plasticity. Sensory neurons in the ventral-caudal cluster of the pleural ganglion were immunoreactive for TbetaR-II, as well as many neurons in the pedal, abdominal, buccal, and cerebral ganglia. Sensory neurons cultured in isolation and cocultured sensory and motor neurons were also immunoreactive. TGF-beta1 affected the biophysical properties of cultured sensory neurons, inducing an increase of excitability that persisted for at least 48 hr. Furthermore, exposure to TGF-beta1 resulted in a reduction in the firing threshold of sensory neurons. These results provide further support for the hypothesis that TGF-beta1 plays a role in long-term synaptic plasticity in Aplysia.

Levels of serotonin in the hemolymph of Aplysia are modulated by light/dark cycles and sensitization training.

Serotonin (5-hydroxytryptamine, 5-HT) modulates the behavior and physiology of both vertebrate and invertebrate animals. Effects of injections of 5-HT and the morphology of the serotonergic system of Aplysia indicate that 5-HT may have a humoral, in addition to a neurotransmitter, role. To study possible humoral roles of 5-HT, we measured 5-HT in the hemolymph. The concentration of 5-HT in the hemolymph was approximately 18 nM, a value close to previously reported thresholds for eliciting physiological responses. The concentration of 5-HT in the hemolymph expressed a diurnal rhythm. In addition, electrical stimulation that leads to long-term sensitization significantly increased levels of 5-HT in the hemolymph during training, 1.5 hr after training, and 24 hr after training. Moreover, levels of 5-HT in the hemolymph were significantly correlated with the magnitude of sensitization. The half-life of an increase in 5-HT in the hemolymph was approximately 0.5 hr. Therefore, the persistent increase of 5-HT in the hemolymph 24 hr after sensitization training indicates that training caused a long-lasting increase in the release of 5-HT. This long-lasting increase in 5-HT in the hemolymph was blocked by treatment with an inhibitor of protein synthesis during training. Based on the levels of 5-HT in the hemolymph and its regulation by environmental events, we propose that 5-HT has a humoral role in regulation of the behavioral state of Aplysia. In support of this hypothesis, we found that increasing levels of 5-HT in the hemolymph led to significant alterations in feeding behavior. Increasing levels of 5-HT during the daytime when they were normally low increased the latency to assume feeding posture from daytime to nighttime values.

Aging affects the ocular circadian pacemaker of Aplysia californica.

The eye of Aplysia has been used to explore various aspects of circadian rhythms. The authors discovered that age has profound effects on the circadian rhythm of nerve impulses from the eye. With age, there was a significant decrease in the amplitude of the rhythm. The decrease appeared to be continuous over the life span of the animal and was observed both in vitro and in vivo. The free-running period and phase angle of the rhythm steadily increased with age, indicating that the pacemaker itself was affected by age. Rates of transcription and translation were significantly increased with age, suggesting that age-associated alterations of the pacemaker may occur through changes in macromolecular synthesis. Interestingly, eyes from some older (> or = 10 months) animals had "cloudy" lenses (cataracts). Highly damped or arrhythmic rhythms always were seen in eyes with cloudy lenses. Morphology of eyes with cloudy lenses indicated severe retinal degeneration. No such degeneration was observed in eyes with clear lenses that were used in the analysis of the rhythm with age.

Effects of cyclin-dependent kinase inhibitors on transcription and ocular circadian rhythm of Aplysia.

Cyclin-dependent kinases (CDKs) mediate cell-cycle phase transitions. Recently, CDKs have been associated with non-cell-cycle roles such as DNA repair, transcription, and phosphate metabolism in yeast. The cyclical processes, circadian rhythms and the eukaryotic cell cycle, are similar in many respects. It is possible that a kinase like CDK is involved in the control of circadian rhythms. In this study, the effects of CDK inhibitors (olomoucine, roscovitine, and butyrolactone I) on the Aplysia ocular circadian rhythm were investigated. Continuous treatments with olomoucine (10 microM) lengthened the free-running period of the rhythm, and pulse treatments of olomoucine (6 h, 100 microM) delayed the rhythm. The effects of olomoucine on the rhythm were qualitatively similar to those of a reversible inhibitor of transcription, 5,6-dichloro-beta-1-ribobenzimidazole. Subsequently, olomoucine was found to inhibit RNA synthesis in the eye of Aplysia and Bulla. All of the other CDK inhibitors used in this study also inhibited transcription in the eye of Aplysia, and their effects on transcription correlated with their effects on the circadian rhythm. This study adds substantial evidence to that previously obtained by using 5,6-dichloro-beta-1-ribobenzimidazole for a role of transcription in the mechanism responsible for circadian rhythmicity in the eye of Aplysia. Also, these results indicate that caution is warranted in interpreting results obtained by using CDK inhibitors, because these drugs appear to inhibit transcription as well as CDKs.

Identification of specific mRNAs affected by treatments producing long-term facilitation in Aplysia.

Neural correlates of long-term sensitization of defensive withdrawal reflexes in Aplysia occur in sensory neurons in the pleural ganglia and can be mimicked by exposure of these neurons to serotonin (5-HT). Studies using inhibitors indicate that transcription is necessary for production of long-term facilitation by 5-HT. Several mRNAs that change in response to 5-HT have been identified, but the molecular events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we investigated the effects of 5-HT (1.5 hr) on levels of mRNA in pleural-pedal ganglia using in vitro translation. Four mRNAs were affected by 5-HT, three of which were identified as calmodulin (CaM), phosphoglycerate kinase (PGK), and a novel gene product (protein 3). Using RNase protection assays, we found that 5-HT increased all three mRNAs in the pleural sensory neurons. CaM and protein 3 mRNAs were also increased in the sensory neurons by sensitization training. Furthermore, stimulation of peripheral nerves of pleural-pedal ganglia, an in vitro analog of sensitization training, increased the incorporation of labeled amino acids into CaM, PGK, and protein 3. These results indicate that increases in CaM, PGK, and protein 3 are part of the early response of sensory neurons to stimuli that produce long-term facilitation, and that CaM and protein 3 could have a role in the generation of long-term sensitization.

Role of calcium-calmodulin-dependent protein kinase II in modulation of sensorimotor synapses in Aplysia.

The Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, [1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne) (KN-62), was used to investigate the role of CaMKII in synaptic transmission and serotonin (5-HT)-induced facilitation in Aplysia. Application of KN-62 (10 microM) by itself increased the amplitude of excitatory postsynaptic potentials (EPSPs) at sensorimotor synapses in pleural-pedal ganglia. Moreover, in the presence of KN-62, 5-HT-induced short-term facilitation was attenuated. Application of KN-62 by itself slightly increased the duration of action potentials in isolated sensory neuron somata but did not block spike broadening produced by 5-HT. KN-62 had no effect on excitability of isolated sensory neuron somata nor did it block 5-HT-induced enhancement of excitability. These results indicate that the attenuation of short-term facilitation by KN-62 is not due to modulation of the membrane currents contributing to 5-HT-induced spike broadening or enhancement of excitability. Rather, these data are consistent with the hypothesis that CaMKII contributes to the regulation of sensorimotor connections and that it has a role in spike-duration-independent processes contributing to short-term facilitation.

Identification of two phosphoproteins affected by serotonin in Aplysia sensory neurons.

Protein phosphorylation appears to play important roles in the mechanisms responsible for presynaptic facilitation in Aplysia. To screen for phosphoproteins that may be involved in facilitation, we previously examined protein phosphorylation in pleural sensory neurons as a function of different durations (2 min, 25 min and 1.5 h) of serotonin treatments. Different durations of serotonin had unique effects on the phosphorylation of different sets of proteins. To determine the functions of these phosphoproteins, we have begun to obtain their amino acid sequences using protein microsequencing techniques. We report here partial sequencing of 2 such proteins. One protein (S6), whose phosphorylation was affected by 2 min treatments with serotonin, appeared to be an intermediate filament protein. Another protein (L55), whose phosphorylation was affected by 1.5-h treatments with serotonin, appeared to be a calmodulin-like Ca(2+)-binding protein. Although the exact cellular functions for S6 and L55 are not known, obtaining partial sequences of these proteins sets the stage for future studies that will examine their regulation and their specific roles in facilitation.

Role of transforming growth factor-beta in long-term synaptic facilitation in Aplysia.

The role of transforming growth factor-beta (TGF-beta) in long-term synaptic facilitation was examined in isolated Aplysia ganglia. Treatment with TGF-beta1 induced long-term facilitation (24 and 48 hours), but not short-term (5 to 15 minutes) or intermediate-term (2 to 4 hours) facilitation. The long-term effects of TGF-beta1 were not additive with those of serotonin. Moreover, serotonin-induced facilitation was blocked by an inhibitor of TGF-beta. Thus, activation of TGF-beta may be part of the cascade of events underlying long-term sensitization, consistent with the hypothesis that signaling molecules that participate in development also have roles in adult neuronal plasticity.

A developmental gene (Tolloid/BMP-1) is regulated in Aplysia neurons by treatments that induce long-term sensitization.

Long-term sensitization training, or procedures that mimic the training, produces long-term facilitation of sensory-motor neuron synapses in Aplysia. The long-term effects of these procedures require mRNA and protein synthesis (Montarolo et al., 1986; Castellucci et al., 1989). Using the techniques of differential display reverse transcription PCR (DDRT-PCR) and ribonuclease protection assays (RPA), we identified a cDNA whose mRNA level was increased significantly in sensory neurons by treatments of isolated pleural-pedal ganglia with serotonin for 1.5 hr or by long-term behavioral training of Aplysia. The effects of serotonin and behavioral training on this mRNA were mimicked by treatments that elevate cAMP. The aplysia mRNA increased by serotonin and behavioral training was 41-45% identical to a developmentally regulated gene family which includes Drosophila tolloid and human bone morphogenetic protein-1 (BMP-1). Both tolloid and BMP-1 encode metalloproteases that might activate TGF-beta (transforming growth factor beta)-like molecules or process procollagens. Aplysia tolloid/BMP-1-like protein (apTBL-1) might regulate the morphology and efficacy of synaptic connections between sensory and motor neurons, which are associated with long-term sensitization.

The use of a reversible transcription inhibitor, DRB, to investigate the involvement of specific proteins in the ocular circadian system of Aplysia.

Previously, the effects of 2-h treatments with the reversible transcription inhibitor 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) on the phase of the circadian rhythm in the eye of Aplysia californica were studied. Here we report a study of the effects of DRB on protein synthesis and a more detailed investigation of the effects of DRB on the phase of the circadian rhythm. Treatments of DRB for 30 min reduced the rate of transcription to about 30% of control values, and this inhibition reversed completely within 2 h after the end of the treatment. A phase-response curve was obtained for 30-min treatments of DRB. Shorter (30 min) treatments with DRB produced phase shifts comparable to those produced by treatments with DRB for 2 h. The phase-response curve obtained using 30-min treatments of DRB was similar to one obtained using 2-h treatments with respect to the phase at which DRB exerts its maximum effect on the rhythm (around circadian time [CT] 6). However, some aspects of the two phase-response curves were different. The effect of DRB on the phase of the rhythm appeared rapidly after removal of DRB treatments given during CT 22-6, but the effects of DRB on the phase of the rhythm appeared more slowly (approximately 10 h) after the treatments given during CT 6-12. Because the effects of DRB on the phase of the overt rhythm appear to be rapid at a particular phase, it is very likely that DRB affects the phase of the rhythm by altering the synthesis of proteins during or shortly after the treatment. Thus we searched for proteins whose synthesis was altered by DRB. Incorporation of labeled amino acids into 2 proteins was found to be altered during the DRB treatment, whereas 15 proteins were affected after the DRB treatment. Among the proteins affected during or shortly after the DRB treatment were four previously identified proteins affected by other treatments that can shift the phase of the eye circadian rhythm. These four proteins are worthy of further study as possible candidates for components of the circadian oscillator.

Identification of three proteins in the eye of Aplysia, whose synthesis is altered by serotonin (5-HT). Possible involvement of these proteins in the ocular circadian system.

Previous results using translation inhibitors in the ocular circadian system of Aplysia suggest that protein synthesis may be involved in the light and serotonin (5-HT) entrainment pathways or perhaps in the circadian oscillator. Proteins have been previously identified whose synthesis was altered by treatments of light capable of perturbing the phase of the circadian rhythm in the eye of Aplysia. We extended these studies by investigating the effects of other treatments that perturb the ocular circadian rhythm on protein synthesis. 5-HT altered the synthesis of nine proteins. Interestingly, five of the proteins affected by treatments with 5-HT were previously shown to be affected by treatments with light. Four of the proteins affected by treatments with 5-HT were also affected by treatments with analogs of cAMP, a treatment which mimics the effects of 5-HT on the ocular circadian rhythm. To identify the cellular function of some of these proteins, we obtained their partial amino acid sequences. Based on these sequences and additional characterizations, a 78-kDa, pI 5.6 Aplysia protein appears to be glucose-regulated protein 78/binding protein, and a 36-kDa, pI 5.7 Aplysia protein appears to be porin/voltage-dependent anion channel. Heat shock experiments on Aplysia eyes revealed that yet another one of the Aplysia proteins (70 kDa) affected by 5-HT appears to be a heat-inducible member (heat shock protein 70) of the family of heat shock proteins. These findings suggest that these three identified proteins, together or individually, may be involved in some way in the regulation of the timing of the circadian oscillator in the eye of Aplysia.

Dynamics of protein phosphorylation in sensory neurons of Aplysia.

Protein phosphorylation plays important roles in the mechanisms underlying serotonin (5-HT)-induced presynaptic facilitation of Aplysia sensory neurons. To study mechanisms involved in facilitation, we investigated the pattern of protein phosphorylation in sensory neurons as a function of different durations of 5-HT. Two minutes and 1.5 hr treatments with 5-HT altered the phosphorylation of 5 and 10 proteins, respectively. These different duration treatments with 5-HT produced unique effects on the phosphorylation of different sets of proteins. This result suggests that cells may encode and measure the duration of a stimulus by the pattern of specific proteins that are phosphorylated or dephosphorylated. In addition, because the changes in phosphorylation produced by 2 min treatments with 5-HT were not observed after 25 min treatments with 5-HT, mechanisms must exist for the transient phosphorylation of some proteins even when the 5-HT treatment persists. Anisomycin, an inhibitor of protein synthesis, blocked the effect of 1.5 hr treatments with 5-HT on the phosphorylation of six proteins but had no effect on the phosphorylation change of four other proteins. Both CPT-cAMP (an activator of protein kinase A) and PDAc (an activator of protein kinase C) mimicked the effects of 5-HT on four proteins. Interestingly, the effect of 5-HT on these four proteins did not require protein synthesis. CPT-cAMP, but not PDAc, mimicked the effect of 5-HT on one protein (L55) and, the effect of 5-HT on this protein appeared to require protein synthesis. Because both activation of PKA and protein synthesis are involved in the induction of long-term facilitation, protein L55 is a good candidate for a protein that might play a key role in long-term facilitation. Finally, the effects of 5-HT on four proteins were not mimicked by either CPT-cAMP or PDAc. This finding raises the interesting possibility that some effects of 5-HT are mediated by second-messenger systems other than PKA or PKC.

Effects on protein synthesis produced by pairing depolarization with serotonin, an analogue of associative learning in Aplysia.

A form of associative plasticity in Aplysia, activity-dependent neuromodulation, involves the convergence of neuronal activity and the effects of a modulatory transmitter. To investigate the role of protein synthesis in associative plasticity, we examined the effects of a biochemical analogue of activity-dependent neuromodulation on the level of incorporation of labeled amino acid into proteins. To mimic associative training, abdominal ganglia were exposed to paired treatments of a depolarizing agent, elevated potassium, and a modulatory transmitter, serotonin. The effects of elevated potassium and serotonin applied alone were also examined. At least two proteins (nos. 9 and 17) were affected in a nonadditive way by the paired procedure. Incorporation of label into protein 9 was increased by the paired procedure but was not affected by either elevated potassium or serotonin. Incorporation of label into protein 17 was significantly affected by elevated potassium or serotonin, but the effect of the paired procedure was significantly less than the summed effects of elevated potassium and serotonin applied alone. These results indicate that changes in protein synthesis may be important in the induction of associative plasticities. Amino acid sequences of two peptides derived from protein 9 were obtained. Then, a partial cDNA clone for protein 9 was obtained by performing PCR with degenerate primers corresponding to portions of the sequences of the two peptides. The sequence of protein 9 is related to sequences previously reported for a family of genes comprising the stringent starvation protein of Escherichia coli, auxin-induced proteins of plants, and glutathione S-transferases of a number of organisms.

Identification of an annexin-like protein and its possible role in the Aplysia eye circadian system.

Light and serotonin regulate the phase of the circadian rhythm of the isolated eye of Aplysia. To screen for possible protein components of the eye circadian oscillator, we identified a number of proteins whose synthesis was altered in opposite ways by light and serotonin. The cellular function of one of these proteins was investigated by obtaining a partial amino acid sequence of it and by examining its immunoreactivity. A 38-amino acid sequence was obtained from a 40-kDa (isoelectric point 5.6) protein. A greater than 60% amino acid identity existed between this sequence and sequences of a family of calcium/phospholipid-binding proteins called annexins. Furthermore, the 40-kDa protein reacted with antibodies generated against a conserved amino acid sequence of annexins and with antibodies raised against human annexin I. The identification of the 40-kDa, light- and serotonin-regulated protein as an annexin led us to hypothesize that arachidonic acid metabolism plays a role in the Aplysia eye circadian system. To test this hypothesis, we examined the ability of an inhibitor of the arachidonic acid metabolic pathway to perturb the eye rhythm. Pulse treatments of isolated eyes with a lipoxygenase inhibitor, nordihydroguaiaretic acid, phase shifted the rhythm. The phase-shifting ability of nordihydroguaiaretic acid suggests that arachidonic acid and some of its metabolites may play a role in the eye circadian system. The results of our studies raise the possibility that links may exist between the 40-kDa annexin-like protein, arachidonic acid metabolism, and the circadian oscillator.