Cysteine Sulfoxides Enhance Steroid Hormone Production via Activation of the Protein Kinase A Pathway in Testis-Derived I-10 Tumor Cells.

Testosterone plays an important role in male sexual characteristics and maturation, and decreased testosterone levels increase the risk of several diseases. Recently, onion extract rich in cysteine sulfoxides, which are amino acids unique to onions, has been reported to alleviate age-related symptoms resulting from decreased testosterone levels in males. However, the mechanism underlying the suppression of low testosterone levels by cysteine sulfoxides has not been elucidated. In this study, we found that onion extract containing cysteine sulfoxides enhanced progesterone, a precursor of testosterone, in mouse testis-derived I-10 tumor cells. Furthermore, cysteine sulfoxides activated protein kinase A (PKA) and cyclic adenosine monophosphate response element-binding protein, which are key factors in steroidogenesis. These results suggest that cysteine sulfoxides enhance steroid hormone production via activation of the PKA signaling pathway.

The Yerkes-Dodson law and appropriate stimuli for conditioned taste aversion in Lymnaea.

The pond snail Lymnaea stagnalis can learn conditioned taste aversion and then consolidate it into long-term memory (LTM). A high-voltage electric shock was used as the unconditioned stimulus, where we have previously used KCl. We varied the strength of both the conditioned and unconditioned stimuli to determine whether the so-called Yerkes-Dodson law prevailed. This is an empirical relationship between the state of arousal and LTM formation, showing that there is an optimal level of arousal leading to memory formation. However, too little or too much arousal results in poorer LTM. We found here that the most appropriate stimuli to use in taste aversion training in Lymnaea were a 10 mmol l(-1) sucrose solution as the conditioned stimulus and a 3 s electric shock as the unconditioned stimulus.

Memory block: a consequence of conflict resolution.

Food deprivation for 1 day in the pond snail Lymnaea stagnalis before aversive classical conditioning results in optimal conditioned taste aversion (CTA) and long-term memory (LTM) formation, whereas 5-day food deprivation before training does not. We hypothesize that snails do in fact learn and form LTM when trained after prolonged food deprivation, but that severe food deprivation blocks their ability to express memory. We trained 5-day food-deprived snails under various conditions, and found that memory was indeed formed but is overpowered by severe food deprivation. Moreover, CTA-LTM was context dependent and was observed only when the snails were in a context similar to that in which the training occurred.

Involvement of insulin-like peptide in long-term synaptic plasticity and long-term memory of the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA-LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA-LTM in the Lymnaea CNS.

An increase in insulin is important for the acquisition conditioned taste aversion in Lymnaea.

Conditioned taste aversion (CTA) in Lymnaea is brought about by pairing a sucrose solution (the conditioned stimulus, CS) with an electric shock (the unconditioned stimulus, US). Following repeated CS-US pairings, CTA occurs and it is consolidated into long-term memory (LTM). The best CTA is achieved, if snails are food-deprived for 1 day before training commences. With a longer period of food deprivation (5 days), learning and memory formation does not occur. It has been hypothesized that the levels of insulin in the central nervous system (CNS) are very important for CTA to occur. To test his hypothesis, we injected insulin directly into 5-day food-deprived snails. The injection of insulin, as expected, resulted in a decrease in hemolymph glucose concentration. Consistent with our hypothesis with insulin injection, learning and memory formation of CTA occurred. That is, the 'insulin spike' is more important than an increase in hemolymph glucose concentration for CTA-LTM. If we injected an insulin receptor antibody into the snails before the insulin injection, learning was formed but memory formation was not, which is consistent with our previous study. Therefore, a rise in the insulin concentration (i.e., insulin spike) in the CNS is considered to be a key determining factor in the process of CTA-LTM.

What are the elements of motivation for acquisition of conditioned taste aversion?

The pond snail Lymnaea stagnalis is capable of being classically conditioned to avoid food and to consolidate this aversion into a long-term memory (LTM). Previous studies have shown that the length of food deprivation is important for both the acquisition of taste aversion and its consolidation into LTM, which is referred to as conditioned taste aversion (CTA). Here we tested the hypothesis that the hemolymph glucose concentration is an important factor in the learning and memory of CTA. One-day food deprivation resulted in the best learning and memory, whereas more prolonged food deprivation had diminishing effects. Five-day food deprivation resulted in snails incapable of learning or remembering. During this food deprivation period, the hemolymph glucose concentration decreased. If snails were fed for 2days following the 5-day food deprivation, their glucose levels increased significantly and they exhibited both learning and memory, but neither learning nor memory was as good as with the 1-day food-deprived snails. Injection of the snails with insulin to reduce glucose levels resulted in better learning and memory. Insulin is also known to cause a long-term enhancement of synaptic transmission between the feeding-related neurons. On the other hand, injection of glucose into 5-day food-deprived snails did not alter their inability to learn and remember. However, if these snails were fed on sucrose for 3min, they then exhibited learning and memory formation. Our data suggest that hemolymph glucose concentration is an important factor in motivating acquisition of CTA in Lymnaea and that the action of insulin in the brain and the feeding behavior are also important factors.

Successful wound closure using fibrin glue for intractable neobladder-urethral anastomosis leakage after radical cystectomy and neobladder reconstruction.

Introduction: Complications of cystectomy and neobladder reconstruction such as anastomotic leakage have been reported. It is a common complication; however, most cases improve conservatively. The use of fibrin glue for fistulas has been reported, but no reports have shown its effectiveness for urinary tract anastomotic leakage. We experienced a case of intractable neobladder-urethral anastomosis leakage after radical cystectomy and neobladder reconstruction, which was effectively managed using fibrin glue. Case presentation: A 70-year-old man underwent radical cystectomy and ileal neobladder reconstruction for invasive bladder cancer with urothelial carcinoma. After surgery, the urethral catheter fell off and the anastomotic leakage did not improve by adjusting the position of the urethral catheter and percutaneous nephrostomy. We closed the intractable neobladder-urethral anastomotic leakage by injecting fibrin glue and the leakage completely disappeared. Conclusion: Injecting fibrin glue into anastomotic site can be effective in severe neobladder-urethral anastomosis leakage.

DNA endoreplication in the brain neurons during body growth of an adult slug.

Endoreplication is DNA synthesis without cell division. Giant neurons observed in the brains of mollusks are thought to be generated as a result of DNA endoreplication. It has been hypothesized that neuronal size becomes larger in parallel with an increase in body size and that DNA endoreplication is involved in this process to meet the increasing demand for macromolecules in neurons. There is, however, no experimental evidence for this hypothesis to date. In the present study, we investigated the following quantitatively: (1) the size of the brain and each ganglion, (2) the size of identified neurons, (3) the total number of neurons undergoing DNA endoreplication, (4) the total number of the neurons containing a cardioexcitatory peptide, and (5) the gene expression level per neuron, using terrestrial slugs whose body growth was regulated through the amount of food supplied in the laboratory. The body growth was accompanied by increases in the sizes of both neurons and ganglia and triggered more frequent DNA endoreplication events in each ganglion of the growth-promoted slugs, without increasing the total number of neurons. Increase in the neuronal size also involved the increase in the amount of transcripts expressed in a single neuron. This is the first quantitative evidence showing that the DNA endoreplication, neuronal size, and gene expression are increased concomitantly with body growth in adult mollusks.

Whole genome amplification in large neurons of the terrestrial slug Limax.

The brain of gastropod mollusks contains giant neurons whose nuclei are enlarged with a large amount of genomic DNA. Such DNA is produced by repeated endoreplication. We have previously demonstrated that the frequency of the neuronal DNA endoreplication is correlative to the body growth of the adult land slug and to the increase in the amount of transcripts within the neuron. However, it has long been controversial whether the neuronal DNA endoreplication entails whole genome amplification (polyploidy), or whether only the necessary genomic loci are amplified (polyteny, polysomy, or cis-amplification by unequal recombination). In the present study, we adopted two modern techniques - quantitative genomic PCR and 5'-bromodeoxyuridine labeling - to distinguish between these two possibilities. Our results demonstrated that multiple genomic loci were amplified to the same extent irrespective of the transcriptional activities at these loci. Moreover, the visceral giant cell, the biggest neuron in the slug's brain, was estimated to contain approximately 10 000-times as much genomic DNA as the haploid amount. The 5'-bromodeoxyuridine-labeling experiments also revealed a uniform DNA synthesis within the nucleus. These results strongly support the idea that the giant neurons contain a polyploid genome rather than a locus-specific amplified genome.

Analysis of DNA endoreplication in the brain neurons in the terrestrial slug, Limax valentianus.

DNA endoreplication is the DNA synthesis without cell division, resulting in the generation of a nucleus containing a larger amount of genomic DNA compared to a normal diploid genome. There are many such giant neurons in the molluscan brain that are generated as a result of repeated endoreplication. However, it has been controversial whether the endoreplication is the whole genome replication (polyploidy) or the local amplification of the genes that are necessary for the neuron's function (polyteny/polysomy). Here in this study, we investigated these two possibilities by (1) immunohistochemical analysis of the distribution of 5'-bromodeoxyuridine incorporated into the nuclei of the brain neurons, and by (2) quantitative genomic PCR directed to two different genes expressed in specific brain regions. Our data supported the view that the DNA endoreplication is the whole genome replication rather than the local amplification of a specific genomic region.

Two pairs of tentacles and a pair of procerebra: optimized functions and redundant structures in the sensory and central organs involved in olfactory learning of terrestrial pulmonates.

Terrestrial pulmonates can learn olfactory-aversion tasks and retain them in their long-term memory. To elucidate the cellular mechanisms underlying learning and memory, researchers have focused on both the peripheral and central components of olfaction: two pairs of tentacles (the superior and inferior tentacles) and a pair of procerebra, respectively. Data from tentacle-amputation experiments showed that either pair of tentacles is sufficient for olfactory learning. Results of procerebrum lesion experiments showed that the procerebra are necessary for olfactory learning but that either one of the two procerebra, rather than both, is used for each olfactory learning event. Together, these data suggest that there is a redundancy in the structures of terrestrial pulmonates necessary for olfactory learning. In our commentary we exemplify and discuss functional optimization and structural redundancy in the sensory and central organs involved in olfactory learning and memory in terrestrial pulmonates.

Unilateral memory storage in the procerebrum of the terrestrial slug Limax.

In most sensory modalities, neuronal inputs are bilaterally processed in a higher center. In some animal species, however, functional lateralization is sometimes observed in the sensory processing at the higher level. For the terrestrial slug Limax, olfaction is the most important sensory modality and this slug can acquire odor-aversion memories. Previously, it has been demonstrated in bilateral PC ablation experiments that the procerebrum (PC) is necessary for odor-aversion memory, and that the PC is the memory storage site. On the other hand, it has been hypothesized that only the unilateral PC is used for odor-aversion learning. Here we demonstrated that the number of the slugs with intact memory performance was reduced by approximately 50% when the PC was surgically ablated only unilaterally before or after conditioning. There was no difference in the memory performance of the right vs. the left PC-ablated slugs. However, memory deficit from unilateral PC ablation was not observed when the ipsilateral tentacles were also amputated at the same time. We also showed that there was no lateral memory transfer from one PC to the other, after up to 7 days post-conditioning. Our results demonstrated clearly that either the left or right PC is randomly used for olfactory learning, and that the side of use is determined at the level of the olfactory ascending pathway to the PC.

Upside-down gliding of Lymnaea.

The pond snail Lymnaea stagnalis can often be observed moving upside down on its back just below the surface of the water. We have termed this form of movement "upside-down gliding." To elucidate the mechanism of this locomotion, we performed a series of experiments involving behavioral analyses and microscopic observations. These experiments were designed (1) to measure the speed of this locomotion; (2) to determine whether the mucus secreted from the foot of Lymnaea repels water, thereby allowing the snail to exploit the surface tension of the water for upside-down gliding; and (3) to observe the beating of foot cilia in this behavior. The beating of these cilia is thought to be the primary driving force for upside-down gliding. Our results demonstrate that upside-down gliding is an efficient active process involving the secretion of mucus that floats up to the water surface to serve as a substrate upon which cilia beat to cause locomotion at the underside of the water surface.

Pancreatic-type Acinar Cell Carcinoma of the Stomach Included in Multiple Primary Carcinomas.

BACKGROUND/AIM: Pancreatic-type acinar cell carcinoma (ACC) in the stomach is extraordinarily rare. We pathologically examined two cases with multiple primary carcinomas, including gastric tumors. PATIENTS AND METHODS: Gastric cancer specimens were examined by immunostaining and electron microscopy. RESULTS: Both cases had cancer cells with acinar patterns, resembling pancreatic ACC. The cancer cells in the first case were positive for exocrine markers, including chymotrypsin, lipase and alpha-1 antichymotrypsin (ACT), as well as neuroendocrine markers, including chromogranin A and synaptophysin. The cancer cells in the second case were positive for chymotrypsin and alpha-1 ACT, while being slightly positive for chromogranin A and synaptophysin. Ultrastructurally, cancer cells contained zymogen granules in both cases. The final diagnosis was pancreatic mixed acinar-neuroendocrine carcinoma and pure pancreatic ACC, respectively. CONCLUSION: We confirmed two cases with gastric pancreatic-type ACC included in multiple primary carcinomas. This type of double cancer has not been reported previously.

Effects of serotonin on the heartbeat of pond snails in a hunger state.

Serotonin (5-hydroxytryptamine: 5-HT) is a multimodal transmitter that controls both feeding response and heartbeat in snails. However, the effects of 5-HT on the hunger state are still unknown. We therefore examined the relation among the hunger state, the heartbeat rate and the 5-HT action in food-starved snails. We found that the hunger state was significantly distinguished by the heartbeat rate in snails. The heartbeat rate was high in the food-satiated snails, whereas it was low in the food-starved snails. An increase in 5-HT concentration in the body boosted the heartbeat rate in the food-starved snails, but did not affect the rate in the food-satiated snails. These results suggest that 5-HT application may mimic the change from a starvation to a satiation state normally achieved by direct ingestion of food.

The cholinergic system in the olfactory center of the terrestrial slug Limax.

Acetylcholine plays various important roles in the central nervous system of invertebrates as well as vertebrates. In the olfactory center of the terrestrial slug Limax, the local field potential (LFP) oscillates, and the change in its oscillatory frequency is thought to correlate with the detection of odor that potentially changes an ongoing behavior of the animal. Acetylcholine is known to upregulate the frequency of the LFP oscillation, and is one of the candidates for the neurotransmitters that are involved in such higher cognitive functions. However, there have been no histological data on the cholinergic system in gastropods, nor are there data on the receptors that are responsible for the upregulation of the oscillatory frequency of LFP due to the lack of analytical tools (such as antibodies or cDNA sequence information on cholinergic system-related genes). Here we cloned the cDNAs of choline acetyltransferase (ChAT), acetylcholinesterase, vesicular acetylcholine transporter, and several nicotinic acetylcholine receptors (nAChRs), and investigated their localization in the brain of Limax. We also generated a polyclonal antibody against ChAT to examine its localization, and investigated pharmacologically the involvement of nAChRs in the LFP oscillation. Our data showed: 1) dense distribution of the neurons expressing mRNAs of ChAT and vesicular acetylcholine transporter in the olfactory center; 2) spatially unique expression patterns of different nAChRs in the olfactory center; 3) involvement of nAChRs in the upregulation of the oscillation; 4) localization of ChAT protein in nerve fibers and/or terminals; and 5) the presence of cholinergic nerves in the tentacles.

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax.

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth.

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax.

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. © 2013 Wiley Periodicals, Inc. Develop Neurobiol, 2013.

Increase in cyclic AMP concentration in a cerebral giant interneuron mimics part of a memory trace for conditioned taste aversion of the pond snail.

Conditioned taste aversion (CTA) can be classically conditioned in the pond snail Lymnaea stagnalis and subsequently be consolidated into long-term memory (LTM). The neural trace that subserves CTA-LTM can be summarized as follows: A polysynaptic inhibitory postsynaptic potential recorded in the neuron 1 medial (N1M) cell in the conditioned snails as a result of activation of the cerebral giant cell (CGC) is larger and lasts longer than that in control snails. The N1M cell is ultimately activated by the CGC via the neuron 3 tonic (N3t) cell. That is, the inhibitory monosynaptic inputs from the N3t cell to the N1M cell are facilitated. The N1M and N3t cells are the members of feeding central pattern generator, whereas the CGC is a multimodal interneuron thought to play a key role in feeding behavior. Here we examined the involvement of a second messenger, cAMP, in the establishment of the memory trace. We injected cAMP into the CGC and monitored the potentials of the B3 motor neuron activated by the CGC. B3 activity is used as an index for the synaptic inputs from the N3t cell to the N1M cell. We found that the B3 potentials were transiently enlarged. Thus, when the cAMP concentration is increased in the CGC by taste aversion training, cAMP-induced changes may play a key role in the establishment of a memory trace in the N3t cell.

Redundancy of olfactory sensory pathways for odor-aversion memory in the terrestrial slug Limax valentianus.

Terrestrial slugs have the ability to learn and remember a food odor paired with an aversive stimulus. Olfaction in slugs involves the tips of two pairs of tentacles, the superior and the inferior tentacles. Sensory nerves in both pairs of the tentacles transmit olfactory information to the structure in the CNS, the procerebrum where learning and memory formation occur. We investigated the role of each pair of tentacles in odor-aversion learning, and examined the ability of slugs to recall memory after selective surgical amputation. Our results show that memory formation was not altered by the amputation of either one of the pairs before or after odor-aversion learning, while the odor sensibility of the slugs was maintained. These data suggest that either pair of tentacles is sufficient for the acquisition and retrieval of aversive olfactory memory.