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.

Circadian clock in photoperiodic time measurement: a test of the Bünning hypothesis.

A technique has been developed for effectively separating the direct inductive effect of a light signal from its effect on the phase of the rhythm of sensitivity to photoperiodic induction. With this technique it has been shown that a 75-minute pulse of light per day, when appropriately positioned with respect to the circadian activity cycle of the sparrow Passer domesticus, is sufficient to produce a response normally produced only by long days. The results cannot be interpreted in terms of a requirement of an absolute amount of either darkness or light and offer strong confirmation of Bünning's hypothesis concerning the mechanism of photoperiodic time measurement.

Entrainment of circadian rhythms by sound in Passer domesticus.

The circadian locomotor rhythm of house sparrows was entrained by a sound stimulus. The birds were maintained at a constant temperature in, dim green light. The entraining agent was 4 (1/2) 12 hours of tape-recorded bird song ,played each day. Variations in the response to this stimulus have been correlated with individual variations in free-running period. This is the first clear demonstration that a biological clock can be influenced by sound stimuli.

Adenylate cyclase activation shifts the phase of a circadian pacemaker.

Forskolin, a highly specific activator of adenylate cyclase, produced both delay and advance phase shifts of the circadian rhythm recorded from the isolated eye of the marine mollusk Aplysia. The phase dependence of the response to forskolin was identical to that with serotonin, which also stimulates adenylate cyclase in the eye. The ability of agents to activate adenylate cyclase in homogenates was correlated with their ability to shift the phase of the circadian oscillator. These results along with earlier findings show that adenosine 3',5'-monophosphate mediates the effect of serotonin on the rhythm and regulates the phase of the circadian pacemaker in the eye of Aplysia.

Serotonin shifts the phase of the circadian rhythm from the Aplysia eye.

A putative neurotransmitter, serotonin, may be used to transmit temporal information in the eye of Aplysia, because it can shift the phase of the circadian rhythm of spontaneous optic nerve impulses from the eye and the eye contains a significant quantity of serotonin. Serotonin acts either directly on the cell, or cells, containing the circadian pacemaker or on cells electronically coupled to the pacemaker cells.

Alteration of the phase and period of a circadian oscillator by a reversible transcription inhibitor.

A function for transcription in the mechanism of a circadian oscillator was investigated with the reversible transcription inhibitor 5,6-dichloro-1-beta-D- ribobenzimidazole (DRB). Two-hour treatments with DRB shifted the phase of the circadian rhythm of the isolated eye of Aplysia, and continuous treatments of DRB lengthened the free running period of this rhythm. Camptothecin, an inhibitor of transcription that is structurally unrelated to DRB, had similar effects on the circadian rhythm. These results suggest that transcription may be part of the circadian oscillating mechanism.

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.

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.

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.

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.

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.

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.

Immunocytochemical localization of serotonergic fibers innervating the ocular circadian system of Aplysia.

The isolated eye of the mollusc, Aplysia californica, contains a circadian pacemaker whose phase can be regulated by serotonin. The results of previous biochemical and physiological studies indicate that serotonin is used as a transmitter of circadian information in the eye. Although the effects of serotonin on various physiological processes in the Aplysia eye have been studied, very little is known about the anatomy of the serotonergic innervation. We have examined the innervation of the eye using immunocytochemical methods. Serotonin-immunoreactive processes were observed in the optic nerve, in the accessory optic nerves, in the connective tissue capsule surrounding the eye, and within the eye itself. There appeared to be two sources of serotonergic input to the eye of Aplysia. One set of immunoreactive fibers was contained in the optic nerve and entered the eye in the neuropil region before radiating outward towards the peripheral retina in the layer below the photoreceptor cell bodies. A second serotonin-immunoreactive input to the eye entered from the accessory optic nerves and these fibers formed a dense plexus of fibers in the connective tissue capsule surrounding the eye. Serotonin-immunoreactive fibers from the plexus penetrated the eye and appeared to terminate in the peripheral portion of the retina. No serotonin-immunoreactive cell bodies were observed in the eye, nerves, or connective tissue capsule. These results support the hypothesis that serotonergic fibers innervate the retina of Aplysia and that these fibers travel through two distinct anatomical pathways: the optic nerve and the accessory optic nerves.

Long-term changes in synthesis of intermediate filament protein, actin and other proteins in pleural sensory neurons of Aplysia produced by an in vitro analogue of sensitization training.

Electrical stimulation of peripheral nerves of isolated pleural-pedal ganglia, an in vitro analogue of long-term behavioral training in Aplysia, produced changes in the synthesis of specific proteins in pleural sensory neurons. The changes in incorporation of [35S]methionine into proteins occurring 24 h after electrical stimulation (late) were determined and compared with changes occurring immediately after stimulation (early). Eight proteins were affected 24 h after electrical stimulation. Three of these proteins were also affected immediately after electrical stimulation. Two of the proteins affected late are components of the cytoskeleton. One protein was identified as actin. The other protein was purified from preparative 2D-gels and partial amino acid sequences of 3 peptides derived from this protein were determined. The peptide sequences were found to be identical to those of an Aplysia intermediate filament protein.

Neural and molecular bases of nonassociative and associative learning in Aplysia.

A model that summarizes some of the neural and molecular mechanisms contributing to short- and long-term sensitization is shown in Figure 14. Sensitizing stimuli lead to the release of a modulatory transmitter such as 5-HT. Both serotonin and sensitizing stimuli lead to an increase in the synthesis of cAMP and the modulation of a number of K+ currents through protein phosphorylation. Closure of these K+ channels leads to membrane depolarization and the enhancement of excitability. An additional consequence of the modulation of the K+ currents is a reduction of current during the repolarization of the action potential, which leads to an increase in its duration. As a result, Ca2+ flows into the cell for a correspondingly longer period of time, and additional transmitter is released from the cell. Modulation of the pool of transmitter available for release (mobilization) also appears to occur as a result of sensitizing stimuli. Recent evidence indicates that the mobilization process can be activated by both cAMP-dependent protein kinase and protein kinase C. Thus, release of transmitter is enhanced not only because of the greater influx of Ca2+ but also because more transmitter is made available for release by mobilization. The enhanced release of transmitter leads to enhanced activation of motor neurons and an enhanced behavioral response. Just as the regulation of membrane currents is used as a read out of the memory for short-term sensitization, it also is used as a read out of the memory for long-term sensitization. But long-term sensitization differs from short-term sensitization in that morphological changes are associated with it, and long-term sensitization requires new protein synthesis. The mechanisms that induce and maintain the long-term changes are not yet fully understood (see the dashed lines in Fig. 14) although they are likely to be due to direct interactions with the translation apparatus and perhaps also to events occurring in the cell nucleus. Nevertheless, it appears that the same intracellular messenger, cAMP, that contributes to the expression of the short-term changes, also triggers cellular processes that lead to the long-term changes. One possible mechanism for the action of cAMP is through its regulation of the synthesis of membrane modulatory proteins or key effector proteins (for example, membrane channels). It is also possible that long-term changes in membrane currents could be due in part to enhanced activity of the cAMP-dependent protein kinase so that there is a persistent phosphorylation of target proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Polypeptide secretion from the eye of Aplysia californica.

The possibility of neurosecretory activity by the eye of Aplysia californica has been suggested by morphological findings. An investigation was conducted to determine if evidence for the release of macromolecular material labeled with radioactive amino acids could be obtained. Depolarization of the eye by superfusion with medium containing an elevated potassium concentration resulted in a significant increase of labeled macromolecular material in the superfusate. This potassium-induced release could be blocked by reduction of the calcium concentration in the superfusing medium. The locus of release appears to be the eye proper and not to involve the optic nerve.

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.

Common set of proteins in Aplysia sensory neurons affected by an in vitro analogue of long-term sensitization training, 5-HT and cAMP.

An in vitro analogue of long-term behavioral training in Aplysia was developed to simulate the intensity and timing of shocks delivered to the body wall of animals during sensitization training. The in vitro training analogue consisted of electrical stimulation of peripheral nerves of isolated pleural-pedal ganglia. We found that the in vitro training analogue led to long-term (24 h) changes in the membrane currents of sensory neurons; these changes were similar to those produced by behavioral training. Using two dimensional polyacrylamide gel electrophoresis, we examined early effects on protein synthesis in pleural sensory neurons induced by the training analogue. Incorporation of amino acid into 5 proteins was affected at the end of training. Incorporation of amino acid into some of these proteins was also affected by serotonin (5-HT) and by an analogue of cyclic adenosine monophosphate (cAMP). These results suggest that the effects on protein synthesis that are produced by the in vitro training analogue are mediated, at least in part, by release of 5-HT and an increase in the level of cAMP in the sensory neurons.

The hunt for mechanisms of circadian timing in the eye of Aplysia.

The goals of our research are to understand how circadian oscillations in the eye of Aplysia california are generated and how entraining agents regulate these oscillations. These goals require identification of the molecular components of the oscillator and entrainment pathways as well as elucidation of the biochemical processes by which these components interact with one another. Our experimental strategy entails tracing environmental information along an entrainment pathway until the last component of the pathway is reached. The isolated eye of Aplysia exhibits a circadian rhythm of optic nerve impulses. This rhythm is regulated by at least two entrainment pathways. A photic pathway entrains the rhythm to light-dark cycles and an efferent serotonergic pathway relays neural information from the CNS to the oscillator. Phase shifting by light appears to involve an increase in the levels of cGMP, depolarization, and protein synthesis. Phase shifting by serotonin appears to involve an increase in the levels of cAMP, hyperpolarization, and protein synthesis. The involvement of protein synthesis in the entrainment pathways, together with the findings that brief treatments of inhibitors of protein synthesis phase shift the rhythm and that continuous treatments of these inhibitors alter the period of the rhythm, indicates that translation is part of the oscillator mechanism. Recent evidence indicates that transcription may also be part of the oscillator mechanism. Brief treatments with DRB, a reversible transcription inhibitor, phase shift the rhythm while continuous treatments with DRB lengthen the period of the rhythm. A comparison of the effects of transcription and translation inhibitors on the rhythm indicates that transcription and translation are closely coupled in the eye circadian system. To know the precise role of transcription and translation in the circadian system, it is necessary to identify and then study specific proteins and mRNAs important for circadian timing. To identify putative oscillator proteins (POPs), we have hunted for proteins whose synthesis or phosphorylation was altered by the entraining agents light and 5-HT and by other agents that perturb the circadian rhythm. By exposing eyes to labeled amino acids in the presence of phase-shifting treatments and then using two-dimensional gel electrophoresis to separate proteins, we found eight proteins that may be considered POPs. To elucidate the cellular function of POPs, we have begun to obtain their amino acid sequences. A 40,000, pI 5.6 protein (POP-1) was identified as a member of the lipocortin family of proteins. Lipocortins are Ca(2+)-phospholipid binding proteins whose functions include inhibition of PLA2.(ABSTRACT TRUNCATED AT 400 WORDS)

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.