Enhanced expression of retinoic acid receptor alpha (RARA) induces epithelial-to-mesenchymal transition and disruption of mammary acinar structures.

The early steps of mammary tumorigenesis include loss of epithelial cell polarity, escape from anoikis, and acquisition of proliferative capacity. The genes responsible for these processes are predicted to be early diagnostic markers or new therapeutic targets. Here we tested 51 genes coamplified with ERBB2 in the 17q12-21 amplicon for these tumorigenic activities using an MCF10A 3D culture-based screening system. We found that overexpression of retinoic acid receptor α (RARA) disrupted normal acinar structure and induced epithelial-to-mesenchymal transition (EMT). The mRNA levels of known EMT-inducing factors, including SLUG, FOXC2, ZEB1, and ZEB2, were significantly increased upon RARA overexpression. Knockdown of ZEB1 suppressed the RARA-mediated EMT phenotype. These results suggest that overexpression of RARA enhances malignant transformation during mammary tumorigenesis.

One-trial conditioned taste aversion in Lymnaea: good and poor performers in long-term memory acquisition.

In the majority of studies designed to elucidate the causal mechanisms of memory formation, certain members of the experimental cohort, even though subjected to exactly the same conditioning procedures, remember significantly better than others, whereas others show little or no long-term memory (LTM) formation. To begin to address the question of why this phenomenon occurs and thereby help clarify the causal mechanism of LTM formation, we used a conditioned taste aversion (CTA) procedure on individuals of the pond snail Lymnaea stagnalis and analyzed their subsequent behavior. Using sucrose as an appetitive stimulus and KCl as an aversive stimulus, we obtained a constant ratio of ;poor' to ;good' performers for CTA-LTM. We found that approximately 40% of trained snails possessed LTM following a one-trial conditioning procedure. When we examined the time-window necessary for the memory consolidation, we found that if we cooled snails to 4 degrees C for 30 min within 10 min after the one-trial conditioning, LTM was blocked. However, with delayed cooling (i.e. longer than 10 min), LTM was present. We could further interfere with LTM formation by inducing inhibitory learning (i.e. backward conditioning) after the one-trial conditioning. Finally, we examined whether we could motivate snails to acquire LTM by depriving them of food for 5 days before the one-trial conditioning. Food-deprived snails, however, failed to exhibit LTM following the one-trial conditioning. These results will help us begin to clarify why some individuals are better at learning and forming memory for specific tasks at the neuronal level.

Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP-induced morphologic changes.

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP-treated and non-treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP-treated astrocytes after stimulation with ATP by high-performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1-43 imaging, appeared in cyclic AMP-treated astrocytes in a punctiform fashion, but not in non-treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP-induced morphologic changes, and in particular, a v-SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP-treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP-induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP-treated astrocytes in response to ATP released from neighboring neurons and astrocytes.

Taste discrimination in conditioned taste aversion of the pond snail Lymnaea stagnalis.

Conditioned taste aversion (CTA) in the pond snail Lymnaea stagnalis has been widely used as a model for gaining an understanding of the molecular and behavioral mechanisms underlying learning and memory. At the behavioral level, however, it is still unclear how taste discrimination and CTA interact. We thus examined how CTA to one taste affected the feeding response induced by another appetitive food stimulus. We first demonstrated that snails have the capacity to recognize sucrose and carrot juice as distinct appetitive stimuli. We then found that snails can become conditioned (i.e. CTA) to avoid one of the stimuli and not the other. These results show that snails can distinguish between appetitive stimuli during CTA, suggesting that taste discrimination is processed upstream of the site where memory consolidation in the snail brain occurs. Moreover, we examined second-order conditioning with two appetitive stimuli and one aversive stimulus. Snails acquired second-order conditioning and were still able to distinguish between the different stimuli. Finally, we repeatedly presented the conditional stimulus alone to the conditioned snails, but this procedure did not extinguish the long-term memory of CTA in the snails. Taken together, our data suggest that CTA causes specific, irreversible and rigid changes from appetitive stimuli to aversive ones in the conditioning procedure.

Advancement of differentiation of oligodendrocyte progenitor cells by a cascade including protein kinase A and cyclic AMP-response element binding protein.

A transcription factor, cyclic AMP-response element binding protein (CREB), which is phosphorylated by protein kinases (PKA and PKC), is known to be involved in the regulation of oligodendrocyte differentiation. However, it is still unclear whether protein kinase A (PKA) and protein kinase C (PKC) are used simultaneously or at different time points to phosphorylate CREB in oligodendrocytes and whether CREB phosphorylation advances oligodendrocyte differentiation or vise versa. Our previous experiments have shown that in the differentiation process from immature to mature cells, CREB phosphorylation depends on PKC activity and leads to the progression of differentiation. In order to gain a better understanding of the process of differentiation from progenitor to immature cells, we identified which protein kinase, i.e., PKA or PKC, regulates CREB phosphorylation and we determined whether CREB phosphorylation advances differentiation or the reverse. Our results showed that CREB phosphorylation is principally regulated by PKA activity in progenitor cells but not by PKC activity, and that this phosphorylation advances the differentiation of progenitor cells to immature cells in oligodendrocytes.

Differentiation of immature oligodendrocytes is regulated by phosphorylation of cyclic AMP-response element binding protein by a protein kinase C signaling cascade.

Previous experiments showed that the expression and phosphorylation levels of cyclic AMP-response element binding protein (CREB) are important factors that regulate oligodendrocyte differentiation. The present study was designed to determine whether CREB phosphorylation advances oligodendrocyte differentiation or vice versa and to identify the protein kinase that primarily regulates CREB phosphorylation. We examined the expression and phosphorylation levels of CREB in developing oligodendrocytes at a specific differentiation stage by double-immunocytochemical staining with specific differentiation markers and antibody for phosphorylated CREB. We found that the CREB expression level increased along oligodendrocyte differentiation, and that its phosphorylated level was highest in immature oligodendrocytes. We also showed that CREB phosphorylation was regulated principally by protein kinase C (PKC) activity in immature oligodendrocytes. Our findings suggest that CREB phosphorylation is dependent on a PKC signaling cascade, and this phosphorylation activates CREB-mediated transcription and advances the differentiation of immature to mature oligodendrocytes.

Nitric oxide synthase and soluble guanylyl cyclase underlying the modulation of electrical oscillations in a central olfactory organ.

We have isolated and characterized the cDNAs for nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) from the terrestrial slug Limax marginatus, and examined the presence and distribution of their mRNAs in the central nervous system using histological techniques and a reverse transcription-polymerase chain reaction method. Our results showed that both bursting and nonbursting neurons in the procerebral lobes contain the mRNAs for both NOS and sGC. We further found that the oscillation frequency of electrical activity in the procerebral lobes increases with increasing intracellular concentrations of cyclic GMP (cGMP). Taken together with previous data on the NO-induced cGMP-like immunoreactivity and on the anatomical distribution of neurites and the localization of synapses of bursting and nonbursting neurons, our present results suggest that NO-induced changes in cGMP concentration modulate the oscillation frequency in the procerebral lobes by acting on the olfactory input pathways, but possibly not on the output pathways, in slugs. .