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This chapter presents a case diagnosed as adenocarcinoma arising from gastric heterotopic pancreas and describes the role of carbohydrate antigen (CA) 19–9 in immunohistochemical staining and its diagnostic significance and limited use in the diagnostic procedures in patients with pancreatic carcinomas. Because there is no antibody available as immunohistochemical (IHC) marker specific to pancreatic neoplastic cells, IHC testing is of limited diagnostic value. Some CAs, such as CA 19–9, appear to have great value as pancreatic tumor markers. They have served (1) to identify that 86% of malignant tumors express the CA 19–9 antigen for patient follow-up and (2) as the predictor of patient response over the course of treatment by measuring the kinetics of serum CA 19–9 concentrations. Use of the CA 19–9 assay in combination with other tumor markers, medical imaging and cytohistologic studies increases the diagnostic rate for pancreatic cancer. The clinical application of genetic analyses specific for pancreatic malignancy is a promising approach both for establishing an accurate diagnosis and for the understanding of novel characteristics of pancreatic cancer.

The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RT-PCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [(14)C]mannitol and [(14)C]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.

The F9 murine embryonal carcinoma cell line provides an attractive system for studying epithelial differentiation and antiproliferative processes. We have recently established F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha and shown that HNF-4alpha triggers the gene expression of tight-junction molecules, occludin, claudin-6, and claudin-7, as well as formation of functional tight junctions and polarized epithelial morphology (Exp. Cell Res. 286, [2003] 288). Since these events were very similar to those induced by retinoids, we investigated whether HNF-4alpha, like retinoid receptors, was involved in the control of cell proliferation. We herein show that HNF-4alpha up-regulates expression of the p21 gene, but not the p15, p16, p18, p19, or p27 gene, in a p53-independent manner, and inhibits cell growth in F9 cells. Similar results were observed in rat lung endothelial cells, in which expression of HNF-4alpha is conditionally induced by doxycycline. Furthermore, we demonstrate, by reporter assay, that HNF-4alpha significantly elevates the transcriptional activity of the p21 promoter. Since, HNF-4alpha is expressed not only in the liver but also in organs containing epithelial cells, such as kidney, intestine, pancreas, and stomach, it might also play critical roles in the regulation of epithelial morphogenesis and proliferation in these organs.

To determine whether wire microelectrodes implanted in the optic disc can be used to elicit cortical potentials.Two or four platinum wire electrodes of two types, viz., the cut-end type and the exposed-tip type, were inserted through the vitreous and fixed in the optic disc of 16 rabbit eyes. Electrically evoked potentials (EEPs) were recorded after bipolar electrical stimulation with the two wire electrodes and by different combinations of the four-electrode system. The optic discs were examined histologically after the experiment.The wire electrodes were successfully implanted and fixed into different positions of the optic disc without serious complications in all 16 eyes. EEPs could be elicited after bipolar electrical stimulation of the optic nerve using either the two-electrode system or different pairs of the four-electrode system. Threshold charge densities to elicit EEPs were 0.32-0.64 mC/cm(2) in eyes using the cut-end type of electrodes and 0.93-6.21 muC/cm(2) in eyes using the exposed-tip type. The amplitude of the EEPs increased with increasing electrical stimulus intensities. Histological evaluation revealed limited damage to the neural tissue adjacent to the electrode track.The visual cortex can be activated by direct microelectrical stimulation of the optic nerve. The acute implantation of the wire microelectrodes into the optic disc by a transvitreal approach is feasible and results in only limited damage to the optic nerve.

We present a case of primitive neuroectodermal tumor (PNET) showing a unique differentiation phenotype based on ultrastructural observation. Rapidly growing tumor involving the retroperitoneum of a 68-year-old woman was characterized by histological findings including proliferation of diffuse poorly differentiated small round cells with scattered rosette formation similar to Homer–Wright type and by the ultrastructure demonstrating cytoplasmic neurosecretary granules and short cytoplasmic cilial structures. These cells revealed immunoreactivity only with neuron-specific enolase (NSE). We also observed a chromosomal translocation, t (11; 22) (q24; q12), which is an identical recurrent alteration found in the neoplastic cells in the spectrum of PNETs. These findings may support the explanation that tumor cells observed in our case shared phenotypes of both neuronal and ependymal cell lineages and give a unique insight suggesting the possible histogenesis of PNETs.

Capillary hemangiomas of the rectum are rare, especially small capillary hemangiomas. We experienced a case of small capillary hemangioma of the rectum, about 2mm in diameter, presenting with massive bleeding. A 51-year-old woman was seen at the hospital because of massive rectal bleeding. There was a previous history of having an internal hemorrhoid. It was difficult to stop the bleeding and an emergency operation was carried out. During the operation we found a white small elevated lesion of about 2mm in diameter locating about 1cm oral side from the dentate line and arterial bleeding from its center. The elevated lesion was excised and sutured. The excised lesion revealed nodular growth pattern of papillary vessels. It was pathologically diagnosed with capillary hemangioma.

To investigate whether electrical stimulation of the optic nerve can elicit an electrical evoked potential (EEP) in rabbits and to determine whether such stimulation is a useful approach for the placement of a visual prosthesis. Two needle-type electrodes were inserted into the optic nerve using a transvitreal approach. For electrical stimulation, monophasic and biphasic pulses were used. By stimulating the optic nerve, the EEP was elicited. After evaluation of the EEP, a histological study was carried out. When electrical stimulation was applied, the EEP could be recorded. The threshold with monophasic and biphasic stimulation was 10 ± 0 µA and 20 ± 8.2 µA, respectively. Histological examination revealed no major complications, such as bleeding or degeneration, which might have resulted from the insertion of electrodes or the electrical stimulation. Electrical stimulation of the optic nerve can elicit an EEP, suggesting that this approach may be useful for a visual prosthesis system. Jpn J Ophthalmol 2004;48:552–557 © Japanese Ophthalmological Society 2004

To know the function of the Ca2+ channel containing alpha(1)2.3 (alpha1E) subunit (Ca(v)2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Ca(v)2.3 channel. The averaged rising rates of Ca2+ transients induced by alpha-D-mannose-bovine serum albumin in the head region of Ca(v)2.3-/- sperm were significantly lower than those of Ca(v)2.3+/+ sperm. A computer-assisted sperm motility assay revealed that straight-line velocity and linearity were greater in Ca(v)2.3-/- sperm than those in Ca(v)2.3+/+ sperm. These results suggest that the Ca(v)2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.

We investigated the role of the Ca v 2.3 (α 1E ) channel in ischemic neuronal injury using Ca v 2.3 mutant mice. In focal ischemia model with a complete occlusion of the middle cerebral artery in vivo, infarct at 24 h was significantly larger in Ca v 2.3 mutant mice compared with that in wild-type controls. In vitro Ca 2+ imaging studies using hippocampal slices revealed that oxygen-glucose deprivation induced a [Ca 2+ ] i increase in the hippocampal CA1 region more vigorously in Ca v 2.3 mutant mice than in wild-type controls, and that tetrodotoxin or bicuculline application abolished the difference between the genotypes. These results suggest that the Ca v 2.3 channel plays a protective role in ischemic neuronal injury by a mechanism in which GABAergic neuronal actions are involved.

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca(2+) signaling. To characterize Ca(2+) signaling in striatal cells, spontaneous cytoplasmic Ca(2+) transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca(2+) transients (referred to as slow Ca(2+) oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca(2+) oscillation. Depletion of the intracellular Ca(2+) store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca(2+) oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca(2+) oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca(2+) oscillation in both putative-neurons and astrocytes. The slow Ca(2+) oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca(2+) oscillation may involve in the neuron-glia interaction in the striatum.

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.

Abnormal CAG repeat expansion in the alpha1A voltage-dependent calcium channel gene is associated with spinocerebellar ataxia type 6, an autosomal dominant cerebellar ataxia with a predominant loss of the Purkinje cell. A reverse transcriptase-polymerase chain reaction analysis of mRNA from mouse Purkinje cells revealed a predominant expression of the alpha1A channel lacking an asparagine-proline (NP) stretch in the domain IV (alpha1A(-NP)). Human alpha1A channels carrying various polyglutamine length with or without NP were expressed in HEK293 cells, and channel properties were compared using a whole-cell voltage clamp technique. alpha1A(-NP), corresponding to P-type channel, with 24 and 28 polyglutamines found in patients showed the voltage dependence of inactivation shifting negatively by 6 and 11 mV, respectively, from the 13 polyglutamine control. Contrarily, the alpha1A channel with NP (alpha1A(+NP)), corresponding to Q-type channel, with 28 polyglutamines exhibited a positive shift of 5 mV. These results suggest that altered function of alpha1A(-NP) may contribute to degeneration of Purkinje cells, which express predominantly alpha1A(-NP), due to the reduced Ca(2+) influx resulting from the negative shift of voltage-dependent inactivation. On the other hand, other types of neurons, expressing both alpha1A(-NP) and alpha1A(+NP), may survive because the positive shift of voltage-dependent inactivation of alpha1A(+NP) compensates Ca(2+) influx.

α 1 subunit of the voltage-dependent Ca 2+ channel is essential for channel function and determines the functional specificity of various channel types. α 1E subunit was originally identified as a neuron-specific one, but the physiological function of the Ca 2+ channel containing this subunit (α 1E Ca 2+ channel) was not clear compared with other types of Ca 2+ channels because of the limited availability of specific blockers. To clarify the physiological roles of the α 1E Ca 2+ channel, we have generated α 1E mutant (α 1E −/−) mice by gene targeting. The lacZ gene was inserted in-frame and used as a marker for α 1E subunit expression. α 1E −/− mice showed reduced spontaneous locomotor activities and signs of timidness, but other general behaviors were apparently normal. As involvement of α 1E in pain transmission was suggested by localization analyses with 5-bromo-4-chloro-3-indolyl β- d -galactopyranoside staining, we conducted several pain-related behavioral tests using the mutant mice. Although α 1E +/− and α 1E −/− mice exhibited normal pain behaviors against acute mechanical, thermal, and chemical stimuli, they both showed reduced responses to somatic inflammatory pain. α 1E +/− mice showed reduced response to visceral inflammatory pain, whereas α 1E −/− mice showed apparently normal response compared with that of wild-type mice. Furthermore, α 1E −/− mice that had been presensitized with a visceral noxious conditioning stimulus showed increased responses to a somatic inflammatory pain, in marked contrast with the wild-type mice in which long-lasting effects of descending antinociceptive pathway were predominant. These results suggest that the α 1E Ca 2 + channel controls pain behaviors by both spinal and supraspinal mechanisms.

Purpose To determine whether retinoic acid (RA)-mediated inhibition of deregulated calpains had any effect on the development of cataract given that accumulating evidence has demonstrated a possible relationship between cataractogenesis and inappropriate activation of calpains. Methods The authors examined for Ca(2+) influx and expression alteration of calpains in F9 cells with or without RAs, such as all-trans retinoic acid (ATRA), and specific stimulant of retinoic acid receptor alpha (RARalpha; Am580) in the presence of oxidative stress, such as mediated by H(2)O(2). They next examined the clinical relevance of RAs by applying these agents to a murine diabetic cataract and observed the development of the disease. Results F9 cells constitute a well-established autonomous cell model for investigating retinoid signaling, partially representing the lens epithelial phenotype, as determined by the expression of aquaporin 0, a specific differentiation marker for lens cells. Treatment with ATRA and Am580 significantly decreased the influx of Ca(2+) into the cells, causally resulting in decreased mRNA expression and inhibited activation of calpains. In addition, RARalpha agonists significantly abrogated the upregulation of calpain 2 induced by H(2)O(2), which is a potential etiological contributor to the diabetic cataract, whereas H(2)O(2) had no effect on calpain 1. Importantly, this RA-mediated gene-expression alteration was sufficient for dramatically inhibiting the development of lens opacity in mice with diabetes. Conclusions Results showed that a certain type of RA inhibits Ca(2+) elevation and subsequent overactivation of calpains, suggesting the potential feasibility of calpain-targeting therapies mediated by RA for cataract.

Glial cell line–derived neurotrophic factor (GDNF) belongs to the neurotrophic factor family. GDNF functions are not restricted to neurons but are also implicated in glial cell development (1). We observed high expression levels of GDNF receptor family α-component 2 (GFRα2) in epiretinal membranes (ERMs) in proliferative diabetic retinopathy (PDR), indicating the involvement of GFRα2 in ERM formation in PDR (2). Here, we examined the vitreous of patients with PDR for the presence of GDNF. We assayed GDNF levels in …

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed “IR-mediated neuronal activation,” and used it for selective stimulation of NE neurons in the locus coeruleus (LC).Drosophila melanogasterIR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations andin vivoelectrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and β-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENTNorepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC–amygdala pathway plays an important role in the recall of taste associative memory.

Ca2+clearance in frog motor nerve terminals was studied by fluorometry of Ca2+indicators. Rises in intracellular Ca2+([Ca2+]i) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca2+transient) reached a peak or plateau within 6–20 stimuli and decayed at least in three phases with the time constants of 82–87 ms (81–85%), a few seconds (11–12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca2+pumps at the cell membrane by external Li+and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li+alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca2+transients. Blocking Ca2+pumps at Ca2+stores by thapsigargin did not affect 100 stimuli-induced Ca2+transients but increased the initial decay time constant of 200 stimuli-induced Ca2+transients with no change in other parameters. Inhibiting mitochondrial Ca2+uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca2+transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca2+transients against [Ca2+]iyielded the supralinear [Ca2+]idependence of Ca2+efflux out of the cytosol. Blocking Ca2+extrusion or mitochondrial Ca2+uptake significantly reduced this [Ca2+]i-dependent Ca2+efflux. Thus Ca2+-dependent mitochondrial Ca2+uptake and plasmalemmal Ca2+extrusion clear out a small Ca2+load in frog motor nerve terminals, while thapsigargin-sensitive Ca2+pump boosts the clearance of a heavy Ca2+load. Furthermore, the activity of plasmalemmal Ca2+pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.