Development and external validation of a nomogram for overall survival after curative resection in serosa-negative, locally advanced gastric cancer.

BACKGROUND: Few nomograms can predict overall survival (OS) after curative resection of advanced gastric cancer (AGC), and these nomograms were developed using data from only a few large centers over a long time period. The aim of this study was to develop and externally validate an elaborative nomogram that predicts 5-year OS after curative resection for serosa-negative, locally AGC using a large amount of data from multiple centers in Japan over a short time period (2001-2003). PATIENTS AND METHODS: Of 39 859 patients who underwent surgery for gastric cancer between 2001 and 2003 at multiple centers in Japan, we retrospectively analyzed 5196 patients with serosa-negative AGC who underwent Resection A according to the 13th Japanese Classification of Gastric Carcinoma. The data of 3085 patients who underwent surgery from 2001 to 2002 were used as a training set for the construction of a nomogram and Web software. The data of 2111 patients who underwent surgery in 2003 were used as an external validation set. RESULTS: Age at operation, gender, tumor size and location, macroscopic type, histological type, depth of invasion, number of positive and examined lymph nodes, and lymphovascular invasion, but not the extent of lymphadenectomy, were associated with OS. Discrimination of the developed nomogram was superior to that of the TNM classification (concordance indices of 0.68 versus 0.61; P < 0.001). Moreover, calibration was accurate. CONCLUSIONS: We have developed and externally validated an elaborative nomogram that predicts the 5-year OS of postoperative serosa-negative AGC. This nomogram would be helpful in the assessment of individual risks and in the consideration of additional therapy in clinical practice, and we have created freely available Web software to more easily and quickly predict OS and to draw a survival curve for these purposes.

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.

Recovery of learning ability after the ablation of the procerebrum in the terrestrial slug, Limax valentianus.

The procerebrum (PC) is indispensable for odor-aversion learning in Limax. On the other hand, the central nervous system (CNS) of some Pulmonata shows robustness against injury, recovering from nerve injury both at the histological and functional levels. To investigate whether the PC of Limax also shows robustness against nerve injury, we tested whether or not the slugs can acquire and retrieve odor-aversion memory after a long recovery period from PC ablation. When the recovery period is short (7 days), the PC-ablated slugs failed to avoid the conditioned odor. But when the recovery period is long (1 month), the PC-ablated slugs successfully avoided the conditioned odor. These results indicate that the CNS including the PC can recover from injury at least at the functional level.

CDP-choline: 1,2-diacylglycerol cholinephosphotransferase from rat liver microsomes. II. Photoaffinity labeling by radioactive CDP-choline analogs.

Photoaffinity labeling of cholinephosphotransferase from rat liver microsomes directly by its substrate, [32P]CDP-choline or by a synthetic photoreactive CDP-choline analog, 3'(2')-O-(4-benzoyl)benzoyl [32P]CDP-choline (BB-[32P]CDP-choline), was examined for the possible identification of its molecular form on subsequent SDS-PAGE followed by 32P-autoradiography. When the partially purified cholinephosphotransferase was photoirradiated in the presence of [32P]CDP-choline, a considerable amount of 32P-radioactivity was incorporated into the TCA-insoluble component. This incorporation was dependent on irradiation time, Mg2+ or Mn(2+)-requiring and inhibited strongly by the presence of Ca2+. Either CDP-choline or CDP-ethanolamine inhibited the ultraviolet irradiation-dependent incorporation of 32P-radioactivity into the TCA-insoluble component in a dose-dependent manner, whereas neither phosphocholine or 5'-CDP had any effect on this process. These results strongly suggested that the observed 32P-incorporation from [32P]CDP-choline into the protein component could be a consequence of the covalent interaction between cholinephosphotransferase and its substrate, [32P]CDP-choline. Two polypeptides, 25 kDa and 18 kDa, with high 32P-radioactivity were clearly identified on a SDS gel after the direct photoaffinity labeling with [32P]CDP-choline for more than 5 min of ultraviolet irradiation. On the other hand, when BB-[32P]CDP-choline was used as a photoaffinity ligand, a single polypeptide with apparent molecular size of 55 kDa could be rapidly photolabeled within 2.5 min, then this band gradually lost its 32P-radioactivity with increasing time of ultraviolet irradiation. Thus, the overall results strongly indicated that cholinephosphotransferase in rat liver microsomes exists most likely as a 55 kDa polypeptide (or subunit) and that 25 kDa and 18 kDa peptides identified after the direct photoaffinity labeling with [32P]CDP-choline were probably the photo-cleavage products of cholinephosphotransferase during the prolonged ultraviolet irradiation, both of which could contain the catalytic domain of the original enzyme protein(s).

Interleukin-12 protects thermally injured mice from herpes simplex virus type 1 infection.

Severe burn injury is associated with increased susceptibility to severe herpesvirus infections. Type 2 cytokines [interleukin (IL)-4 and IL-10] released from burn-associated CD8+ type 2 T cells (BA-type 2 T cells) have been shown to play a role in the increased susceptibility of thermally injured mice (TI-mice) to herpes simplex virus type 1 (HSV-1) infection. Because IL-12 has been shown to inhibit the generation of type 2 T cells, murine rIL-12 was injected into TI-mice exposed to HSV-1 to determine whether IL-12 could influence HSV-1 infections in individuals bearing type 2 T cells. rIL-12 improved the resistance of TI-mice or mice inoculated with T6S cells (a BA-type 2 T cell clone) against HSV-1 infection. Type 2 cytokines were detected in sera of TI-mice or mice inoculated with T6S cells (T6S-mice). However, treatment of TI-mice or T6S-mice with rIL-12 inhibited type 2 cytokine production in the sera of these mice. All TI-mice exposed to a lethal dose of HSV-1 survived when they were treated with a mixture of monoclonal antibodies (mAbs) against type 2 cytokines. Staphylococcal enterotoxin A [an interferon-gamma (IFN-gamma) inducer] stimulated serum IFN-gamma production in TI-mice and T6S-mice treated with rIL-12, whereas no IFN-gamma was produced in mice treated with saline. These results suggest that IL-12 has the potential to protect TI-mice infected with a lethal dose of HSV-1 via a shift to type 1 T cell responses from type 2 T cell responses.

A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody.

Signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. For the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. We have applied this method for screening kinases which phosphorylate STAT3 at serine(727). In this screening, antibody (PS727 antibody) specifically recognizing STAT3 in which serine(727) is phosphorylated was first prepared. Escherichia coli, bacteria expressing a serine(727)-containing fragment of STAT3 which was fused to glutathione-S-transferase (GST) (GST-STAT3-WT) were infected by lambda phage cDNA expression libraries. Phosphorylation of GST-STAT3-WT was effectively performed in E. coli as expected, and clones positive for PS727 antibody immunoreactivity were selected. Isolated 53 clones encode four serine/threonine kinases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual specificity Yak1 related kinase (DYRK), dual specificity Yak1 related kinase 2 (DYRK2) and homeodomain interacting protein kinase 2 (HIPK2). These kinases have a potential to phosphorylate serine(727) in STAT3 protein also in mammalian cells. The present method is considered to be applicable in general to isolate kinases.

Approach to maceration mechanism in enzymatic pulping of bast fibers by alkalophilic pectinolytic enzymes produced by Erwinia species.

Tissue maceration was generally elucidated by the action of endo-polygalacturonase and endo-pectate or -pectin lyase (endo-PAL or -PNL). In a process of screening of Erwinia and Pseudomonas strains for enzymatic pulping of pectocellulosic bast fibers, it was found that their PAL productivity was not completely related with defibration activity, i.e., the fact that an E.chrysanthemi strain showed high PAL productivity but possessed rather low defibration activity. Moreover, defibration activity was parallel to the amount of neutral sugars released during pulping. Based on these fact, the maceration or enzymatic pulping of basts was estimated to proceed not only by cleavage of interfiber bonding cause by PAL action but also another factors. Among three possibilities proposed on the maceration mechanism of basts, it was elucidated by a concerted action of PAL and PNL with an aid of xylanase. In addition, a quantitative determinative method of maceration activity toward basts was also presented.

Neuronal activity in the putamen and the globus pallidus of rabbit during mastication.

The pattern of jaw movements is changed during a masticatory sequence from ingestion of food to its deglutition. The masticatory sequence is divided into three distinct stages in the rabbit. However, the neural mechanism involved in the alteration of the masticatory stages is still unknown. This study was designed to determine whether neuronal activity in the putamen and globus pallidus is related to the alteration of the masticatory stages. Fifty-three percent of the recorded neurons showed significant alterations of activity during mastication. Of these neurons, 16% changed their firing frequency throughout the masticatory sequence (sequence-related neurons) and 84% changed their firing frequency with the transition of the masticatory stages (stage-related neurons). The stage-related neurons were classified into two groups based on their neuronal activity patterns observed during mastication, i.e. simple type and complex type. The former are the neurons that were either facilitated or inhibited once during mastication, and the latter are those showing the facilitation or inhibition twice or more during mastication. Complex-type neurons were observed more frequently in the globus pallidus than in the putamen. These results suggest that the basal ganglia is involved in mastication and may related to the transition between the masticatory stages.

Effects of inorganic constituents of saliva on taste responses of the rat chorda tympani nerve.

The effects of saliva on the taste responses of the chorda tympani nerve to the 4 standard chemical stimuli (sucrose, NaCl, HCl, and quinine hydrochloride) and water were investigated in anesthetized rats. When the tongue was adapted to pilocarpine-stimulated whole saliva (pH 8.7), the magnitude of neural response to sucrose was about 2 times that obtained when the tongue was adapted to distilled water. Under saliva-adapted conditions, the magnitude of responses to other taste stimuli was reduced by 10-30%, and the water response appeared. These changes were dependent on the concentration of electrolytes (Na+, K+, Cl-, and HCO3-) and on the pH of the saliva. When the tongue was adapted to 10-30 mM NaHCO3 (pH 8.4-8.6), taste and water responses were similar to those under saliva-adapted conditions. Single fiber analyses revealed that the enhancement of the sucrose response after adaptation to NaHCO3 was produced by an increased overall activity of sucrose-responsive fibers. The correlation coefficients of the magnitude of the taste responses between the 4 taste stimuli remained unchanged, but the water response showed a high correlation to HCl and quinine hydrochloride responses after adaptation. Possible mechanisms for the effects of saliva on taste and water responses were discussed.

Propagation of synchronous burst discharges from entorhinal cortex to morphologically and electrophysiologically identified neurons of rat lateral amygdala.

Intracellular and field potential recordings were taken from the lateral nucleus of the amygdala in a rat horizontal brain slice preparation that included hippocampal formation. Pyramidal cells comprised the majority of labeled cells (77%). Electrophysiological classification based on hyperpolarizing or depolarizing afterpotentials subdivided both the pyramidal and non-pyramidal cell classes, although pyramidal cells tended to have hyperpolarizing afterpotentials (70%) and non-pyramidal cells tended to have depolarizing afterpotentials (63%). Synchronous population bursts were triggered with single extracellular stimuli in the deep layers of entorhinal cortex. These events propagated from deep layers of entorhinal cortex into the lateral nucleus of the amygdala. Latencies were consistent with a direct entorhinal to amygdala projection. Individual lateral nucleus neurons exhibited responses ranging from a long burst response that included an initial period of 200 Hz firing and a tail of gamma frequency firing lasting over 100 ms (grade 1) to an epsp with no firing (grade 4). Half of pyramidal cells responding to events initiated in entorhinal cortex were found to receive epsps strong enough to trigger firing. Only one stellate neuron fired in response to entorhinal stimulation. Excitatory postsynaptic responses included NMDA and non-NMDA receptor mediated components. We demonstrate that synchronous population events can propagate from entorhinal cortex to the lateral nucleus of the amygdala and that pyramidal neurons of the lateral nucleus are more common targets than stellate neurons. We conclude that other synchronous events such as sharp waves and interictal spikes can spread from entorhinal cortex to amygdala in the same manner.

Corticofugal effects on the activity of thalamic taste cells.

To examine corticofugal influences on afferent thalamic cell responses in the gustatory system, effects of cortical conditioning stimuli on the responses of thalamic taste cells to peripheral stimulation were examined in the rat. Two types of excitability change of thalamic cells were observed; one is inhibitory (for about 60 msec) (20%) and the other is inhibitory (for about 10 msec)--facilitatory (for about 60 msec) (40%) to conditioning stimulus applied to the ventral gustatory area, and half of the cells belonging to the latter type also showed a similar pattern with more marked and prolonged excitability changes to conditioning stimulus applied to the dorsal gustatory area. It was suggested that some of the response characteristics of thalamic and cortical taste cells were attributed to the corticofugal feedback loop.

Taste effects of 'umami' substances in hamsters as studied by electrophysiological and conditioned taste aversion techniques.

Behavioral and electrophysiological experiments were performed to examine whether or not the taste of 'umami' substances such as monosodium glutamate (MSG), disodium 5'-inosinate (IMP), and disodium 5'-guanilate (GMP) is really unique in hamsters. When the animals were conditioned to avoid ingestion of MSG (or IMP) or their mixture by pairing its ingestion with an i.p. injection of LiCl, suppression of drinking generalized to IMP (or MSG), GMP, NaCl, and other sodium salts. Suppression of drinking after conditioning to NaCl generalized to MSG, IMP, GMP, and inorganic sodium salts. These learned aversions to umami substances and sodium salts were abolished by bilateral deafferentation of the chorda tympani, but were not affected by destruction of the bilateral glossopharyngeal nerves. The integrated whole-nerve responses of the chorda tympani to MSG, IMP, and NaCl were similar to each other, consisting of the initial dynamic phase and the following tonic phase. Synergism of chorda tympani responses to a mixture of MSG and IMP was not observed. Across-fiber response patterns of the chorda tympani for MSG, IMP, or their mixture were very similar to that for NaCl. Even the high concentrations of umami substances (0.3 M MSG, 0.3 M IMP, and the mixture) did not elicit any detectable responses in the glossopharyngeal nerve. These results suggest that the taste of umami substances is not unique in the hamster, but is similar to that of sodium salts, and is mediated exclusively via the chorda tympani.

Response properties of lateral hypothalamic neurons during ingestive behavior with special reference to licking of various taste solutions.

Activity of 58 single neurons in the lateral hypothalamic area (LHA) was recorded while Wistar male rats were drinking water and various taste solutions in a test box. A cue tone was presented before opening of a shutter for access to a drinking spout. Except 8 neurons which were non-responsive in the present experimental paradigm, 50 neurons were classified into 3 types according to their response properties: (1) 10 neurons changed their activity with arousal state or circadian rhythm, (2) 10 neurons responded to specific sensory stimuli, i.e. 2 were classified as taste-responsive neurons, which responded excitatory to sodium salts, 3 neurons responded to olfactory stimulation, 5 to somatosensory stimulation applied to the perioral region, and (3) the remaining 30 decreased their activity during licking of liquids regardless of their qualities. Besides this classification, activity of 28 of 58 LHA neurons was altered after onset of the cue tone (or before start of licking), i.e. 24 increased their activity (learned anticipatory response), and 3 modulated their tonic activity into burst discharges corresponding to sniffing, and 1 increased its activity in relation to stepping toward the drinking spout. These data suggest that about half of the LHA neurons increased their activity in anticipatory (searching or approaching) periods just before ingestion, and decreased activity in rewarding periods during ingestion of water or sapid solutions.

Effect of salivation on neural taste responses in freely moving rats: analyses of salivary secretion and taste responses of the chorda tympani nerve.

The outer surface of the mammalian taste receptor cell is usually covered with saliva, which may affect the initial process of gustation. To ascertain the interaction between salivation and gustation, salivary secretion from the submandibular and parotid glands and taste responses to the chorda tympani nerve were analyzed in the rat, during grooming, eating, and licking of the four standard taste stimuli (sucrose, NaCl, HCl, and quinine hydrochloride). Regions of the tongue surface bathed by saliva secreted from the each gland were examined, and it was found that: (1) Rats frequently groomed, and the anterior part of the tongue, innervated by the chorda tympani nerve, was usually covered with a mixture of submandibular saliva and substances on the body surface. (2) Licking of acceptable sucrose and NaCl solutions elicited initial phasic and long-lasting tonic taste responses, and did not evoked saliva enough to wash away the stimuli from the oral cavity. Licking of rejectable quinine evoked only a small phasic taste response and was followed by taste rejection behavior, accompanied by maximum salivation which could wash out the stimuli. (3) When taste responses were compared under awake and anesthetized (the tongue adapted to water) condition, sucrose response was larger, while responses to other taste stimuli were smaller under the awake condition. Rise time of the phasic NaCl response was longer under the awake condition. These taste response alterations may reflect the effects of prolonged adaptation of the tongue to the mixture of submandibular saliva and body surface substances, and flow rate of licked taste stimuli on the tongue surface.

Hypoglossal motor nerve activity elicited by taste and thermal stimuli applied to the tongue in rats.

Single unit activity of hypoglossal motor nerve fibers which innervate the tongue muscles was recorded in lightly anesthetized non-decerebrate and acute decerebrate rats. The pattern of responses to taste and thermal stimuli applied to the tongue surface was classified into 4 types. The type 1 response is characterized by short-lasting rhythmic burst discharges, the type 2 consists of both the rhythmic burst and tonic discharges, the type 3 is long-lasting tonic discharges and the type 4 shows short-lasting burst or short-lasting tonic discharges. In non-decerebrate rats, most of the fibers (93%) showed no or a few spontaneous firings. Sucrose and NaCl were the most effective stimulants, and 70-80% of the fibers showed the type 1 response to these stimuli, sucrose and NaCl, and HCl and quinine produced a similar response profile, respectively. In decerebrate rats, however, about 21% of fibers showed a highly regular spontaneous firing (about 30 Hz). Rhythmic burst responses (types 1 and 2) were not induced, and thermal (especially cold) stimulation elicited much better responses than the taste stimuli. HCl and quinine, but not sucrose and NaCl, produced a similar response profile. These characteristic properties of the response in acute decerebrate rats may in part be attributed to inactivation of a 'rhythmic center' in the brain stem.

Noxious tooth pulp stimulation suppresses c-fos expression in the rat hippocampal formation.

Changes in the expression of immediate early gene c-fos by noxious mechanical stimulation to the mandibular incisor pulp of rats were immunohistochemically examined in the hippocampus (Ammon's horn and dentate gyrus) and the retrohippocampus (subiculum, presubiculum, parasubiculum and entorhinal cortex). The highest control levels were found in subiculum, CA1, dentate and deep medial entorhinal cortex. Lower, but substantial levels were present in the other areas. Whereas weak dentinal stimulation caused increases in c-fos expression in some regions which were not statistically significant, strong tooth pulp stimulation caused a bilateral decrease in c-fos expression in every region except contralateral subiculum. These decreases reached statistical significance in superficial layer parasubiculum bilaterally (p<0.01), bilateral CA1 and ipsilateral side of superficial layer of medial entorhinal cortex (p<0.05). We suggest that inhibitory circuitry in hippocampal formation regions may be activated by peripheral noxious somatosensory inputs and this change in activity is accompanied by a change in the expression of the immediate early gene, c-fos.

Neural activity of chorda tympani mechanosensitive fibers during licking behavior in rats.

The chorda tympani nerve, supplying the anterior two-thirds of the tongue, contains gustatory and mechanosensitive afferent fibers. We have analyzed discharge patterns in rats of various fibers recorded from dissected nerve filaments during licking behavior of which 4 were taste-sensitive and 12 mechanosensitive. The incidence of these two types were estimated electrophysiologically under anesthesia and their conduction velocity measured. Recordings in freely moving animals showed that the mechanosensitive fibers innervating the dorsal part of the tongue gave two burst discharges per lick, suggesting that contact of the tongue with the upper incisors and/or lip occurred during tongue protrusion and retraction. The fibers from the tip of the tongue showed one burst discharge per lick, which was the response to contact with a drinking spout. No rhythmical discharges synchronized with lick signals were observed in the fibers from the lateral part of the tongue or the taste-sensitive fibers. Such mechanoreceptor discharges were difficult to detect in recordings from the whole chorda tympani nerve. This masking of responses was due mainly to activation of a small number of mechanosensitive fibers by licking-induced mechanical stimulation. The lubricating action of saliva also decreased mechanoreceptor sensitivity. Despite their small number, the mechanosensitive fibers had axons with faster conduction velocities (larger diameter) than the taste-sensitive fibers. This was probably the reason why dissected nerve bundles more frequently showed mechanical than taste responses in conscious rats.

Prolonged incubation with neuropeptide Y upregulates beta-adrenoceptors yet does not cause supersensitivity of beta-adrenoceptor signaling.

In neonatal rat ventricular myocytes prolonged incubation with 1 microM neuropeptide Y (2 h-48 h) increased beta-adrenoceptor density 16-24% (n = 4--8, all p < 0.05), an effect prevented by pertussis toxin pretreatment. Prolonged incubation with neuropeptide Y had no effect on adenylycylclase activity stimulated by 5'-guanylylimidodiphosphate or (-)-isoprenaline, probably because of a neuropeptide Y-induced decrease in affinity of the beta-adrenoceptor for agonist. Thus, chronic incubation with an inhibitory agonist does not inevitably lead to supersensitivity of the adenylylcyclase pathway.

Taste nerve responses during licking behavior in rats: importance of saliva in responses to sweeteners.

Taste responses of the rat chorda tympani nerve, innervating taste cells on the anterior part of the tongue, were recorded under awake and anesthetic conditions. Responses to licking of taste solutions in conscious animals were compared with those to taste solutions poured onto the tongue, without contamination of saliva, in anesthetized ones. The most noticeable finding was that sweet-tasting substances such as sugars and amino acids elicited larger magnitudes of neural responses (2-4 times) under awake condition than under anesthetic experimental condition. We conclude that a proper ionic composition of mucous substances covering the surface of the taste cell membrane is important in generating good responses to sweeteners as observed in licking rats.

Cataloging altered gene expression during rat hippocampal long-term potentiation by means of differential display.

We have employed mRNA differential display (DD) to generate a catalog of cDNAs whose expression in the hippocampus was regulated during long-term potentiation (LTP) in dentate gyrus of anesthetized rats. DD with 459 combinations of primer pairs revealed that 80 out of approximately 70000 bands displayed showed a reproducible change in their expression level. These cDNAs were categorized into seven groups according to changes in their temporal expression pattern. Some of these cDNAs were induced rapidly, but transiently, after the LTP induction, some induced rapidly and persistently, some induced slowly, and some down-regulated following LTP. This suggests that a complex molecular hierarchy underlies the maintenance of hippocampal LTP.