Differences in responsiveness of mediodorsal thalamic and medial prefrontal cortical neurons to social interaction and systemically administered phencyclidine in rats.

Phencyclidine (PCP) is a psychotomimetic drug that induces schizophrenia-like symptoms in healthy individuals and behavioral abnormalities with corresponding symptoms of schizophrenia in non-human animals. Our previous studies showed that systemically administered PCP produces tonic activation of neurons in the medial prefrontal cortex (mPFC) of rats and that this activation is mainly via excitatory inputs from regions outside the mPFC. Such long-lasting activation of PFC neurons is now considered to be a pivotal factor in PCP-induced behavioral abnormalities. Although our previous study identified the ventral hippocampus as a possible source of the excitatory inputs, it is not the only source innervating the mPFC. Several regions such as the thalamus also have monosynaptic projections to the mPFC. Recently, increased c-fos expression by systemic PCP administration was reported in the mediodorsal nucleus of the thalamus (MD) and the centromedial nucleus of the thalamus (CM), which have strong reciprocal innervations with the mPFC. However, few studies have reported effects of PCP on the firing activity of MD/CM neurons in unanesthetized animals. In the current study in freely moving rats, we examined effects of systemically administered PCP on the spontaneous firing activity of the MD/CM, after identifying the response properties of recorded neurons in social interaction with an unfamiliar partner. About 30% of MD/CM neurons recorded exhibited tonic excitation following systemic PCP administration, whereas only a few neurons (7%) were inhibited by PCP. The proportion of MD neurons activated by systemic PCP administration was about half of that in the mPFC. Although the proportion of neurons responsive to social interaction did not differ between the two regions (40%), neurons activated during social interaction in the mPFC (90%) were more likely to be affected by systemic PCP administration than those in the MD/CM (45%). These results suggest that neurons responsive to social interaction in the mPFC may be differently affected by PCP than those in the MD/CM.

Stimulatory action of mitemcinal (GM-611), an acid-resistant non-peptide motilin receptor agonist, on colonic motor activity and defecation: spontaneous and mitemcinal-induced giant migrating contractions during defecation in dogs.

The aim of this study was to characterize giant migrating contractions (GMCs) during spontaneous defecation in dogs and to investigate the effect of mitemcinal (an orally active and highly acid-resistant motilin receptor agonist) on colonic motility to assess the possibility of using it for the treatment of colonic motility disorders. To assess colonic motility, strain-gauge force transducers were implanted on the gastrointestinal tract of five dogs, and the behaviour of the dogs was monitored with a noctovision-video camera system. The effect of mitemcinal (0, 3, 10 or 30 mg per dog) and sennoside (300 mg per dog) on colonic motility was assessed 24 h after oral administration. During a 39-day period, the starting point of most of the 140 GMCs was between the transverse colon and the descending colon, but some variation was observed. In the daytime, the GMCs originated from somewhat more proximal positions than at night. Mitemcinal caused an increase in the GMC-index (integration of contractile amplitude and duration) and proximal translocation of the GMC starting point, but did not cause an increase in the number of defecations 12 h after administration. Sennoside, however, caused a significant increase in the number of defecations, an increase in the GMC-index, and prolongation of the duration of GMCs. The GMC starting point in the canine colon varied during spontaneous defecation. Mitemcinal was a potent prokinetic drug to mimic a spontaneous defecation compared with sennoside. Mitemcinal evacuates more intestinal luminal contents during the defecation than does sennoside.

Decreased expression level of SH2 domain-containing protein tyrosine phosphatase-1 (Shp1) is associated with progression of chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) is characterized by t(9;22)(q34;q11) and the aberrant expression of the fusion protein Bcr-Abl that leads to constitutive activation of c-Abl kinase. Bcr-Abl plays a major role in the development and progression of CML through chronic, accelerated, and blast phases. The interaction between Bcr-Abl and other oncogenic molecules has been extensively documented. Nonetheless, negative regulatory mechanisms of Bcr-Abl are not completely defined. One major inhibitory pathway is mediated via the SH2 domain-containing protein tyrosine phosphatase Shp1. In the present study, we demonstrate that Shp1 levels are markedly decreased in advanced stage CML patients compared with those in chronic phase. This process was independent of DNA methylation. Furthermore, we did not detect mutations in the Shp1 gene in CML cell lines or patient samples. These data suggest that the decrease in Shp1 in advanced stage CML patients is due to post-transcriptional modifications. Our findings suggest that the decrease in Shp1 expression levels plays a role in the progression of CML. Also, the decrease in Shp1 and subsequently its inhibitory effect on Bcr-Abl could provide an explanation for imatinib resistance seen in advanced stage CML patients.

Postnatal development of the corticothalamic projections from the visual association cortex to the lateralis posterior complex in the cat.

The postnatal development of the corticothalamic projection from the lateral suprasylvian cortex (LS) to the lateral medialis-suprageniculate nucleus (LM-Sg) of the cat thalamus was assessed by means of the anterograde tracer biocytin. In the adult, two types of corticothalamic fibers were found: type I established a network of fine fibers present throughout the LM-Sg, it was characterized by a linear sequence of small (less than 0.5 microm in diameter), single terminal boutons making contact mainly with thin dendrites and/or dendritic spines. Type II, found less frequently, gave off short, side branches near axon terminals and formed clusters of 5-10 large terminal boutons (0.5-1.5 microm in diameter), making contact predominately with medium-sized dendrites and/or vesicle-containing profiles, forming a synaptic glomerulus. At birth (P0), anterogradely-labeled fibers were found in the LM-Sg as in adults. In the early postnatal period (until P6) as well as around the time of eye-opening (P7-P10) to P21, neonatal fibers were largely unbranched many of them having axons tipped with growth cones. Axon terminals containing synaptic vesicles were rarely observed but when present these exhibited considerable variation in their morphological appearance of synapses. Thus, it was not possible to categorize them into the two types of axons which characterize the adult. After P25, terminal swellings bearing a close resemblance to those of type II fibers begin to appear. In this way, the main two corticothalamic fiber types could be identified. These findings demonstrate that significant postnatal changes occur in the synaptology of corticothalamic fibers in the LM-Sg, particularly with the maturation of type II fibers.

Studies of magnesium in congenital long QT syndrome.

We studied the role of magnesium (Mg) in congenital long QT syndrome (LQTS). Twenty-two congenital LQTS patients and 30 control subjects were included in this study. We measured serum Mg (SMg) level and Mg retention (MgR) level, and evaluated the role of Mg (a high MgR level reflects Mg deficiency in the body). The influence of intravenous Mg infusion on Mg level was evaluated. Relatively low SMg level and high MgR level (LQTS:control = 53:33%, p < 0.01) were recognized in congenital LQTS patients, but there was an overlap with controls. Mg supplementation did not shorten QT interval and there was no significant correlation between Mg levels and QTc interval. Patients with syncopal history showed a higher MgR level (syncope (+):syncope (-) = 70:46%, p < 0.01) and intravenous Mg infusion improved Mg deficiency. These results suggest that some (not all) congenital LQTS patients are in a Mg-deficient state, which may be associated with syncope, and Mg supplementation may prevent recurrent syncope in these patients. Because there are several subtypes of congenital LQTS, perhaps with genetic testing Mg deficiency may be identified as a significant cofactor in some forms, whereas in other forms it is not relevant.

Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy.

Whole-cell assays were implemented to search for efflux pump inhibitors (EPIs) of the three multidrug resistance efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN) that contribute to fluoroquinolone resistance in clinical isolates of Pseudomonas aeruginosa. Secondary assays were developed to identify lead compounds with exquisite activities as inhibitors. A broad-spectrum EPI which is active against all three known Mex efflux pumps from P. aeruginosa and their close Escherichia coli efflux pump homolog (AcrAB-TolC) was discovered. When this compound, MC-207,110, was used, the intrinsic resistance of P. aeruginosa to fluoroquinolones was decreased significantly (eightfold for levofloxacin). Acquired resistance due to the overexpression of efflux pumps was also decreased (32- to 64-fold reduction in the MIC of levofloxacin). Similarly, 32- to 64-fold reductions in MICs in the presence of MC-207,110 were observed for strains with overexpressed efflux pumps and various target mutations that confer resistance to levofloxacin (e.g., gyrA and parC). We also compared the frequencies of emergence of levofloxacin-resistant variants in the wild-type strain at four times the MIC of levofloxacin (1 microg/ml) when it was used either alone or in combination with EPI. In the case of levofloxacin alone, the frequency was approximately 10(-7) CFU/ml. In contrast, with an EPI, the frequency was below the level of detection (<10(-11)). In summary, we have demonstrated that inhibition of efflux pumps (i) decreased the level of intrinsic resistance significantly, (ii) reversed acquired resistance, and (iii) resulted in a decreased frequency of emergence of P. aeruginosa strains that are highly resistant to fluoroquinolones.

Requirement of the Pseudomonas aeruginosa tonB gene for high-affinity iron acquisition and infection.

To investigate the contribution of the TonB protein to high-affinity iron acquisition in Pseudomonas aeruginosa, we constructed tonB-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin. The tonB mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin). The tonB inactivation was shown to make P. aeruginosa unable to acquire iron from the transferrin with either siderophore. Introduction of a plasmid carrying the intact tonB gene restored growth of the tonB mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of the tonB mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin. In addition, animal experiments showed that, in contrast to PAO1, the tonB mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals. The in vivo growth ability and lethal virulence were also restored by introduction of the tonB-carrying plasmid in the tonB mutant. These results indicate clearly that the intact tonB gene-and, therefore, the TonB protein encoded by it-is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in P. aeruginosa and suggest that the TonB-dependent iron acquisition may be essential for P. aeruginosa to infect the animal host.

Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice.

Pseudomonas aeruginosa produces siderophores, pyoverdin and pyochelin, for high-affinity iron uptake. To investigate their contribution to P. aeruginosa infections, we constructed allelic exchange mutants from strain PAO1 which were deficient in producing one or both of the siderophores. When inoculated into the calf muscles of immunosuppressed mice, pyochelin-deficient and pyoverdin-deficient mutants grew and killed the animals as efficiently as PAO1. In contrast, the pyochelin- and pyoverdin-deficient (double) mutant did not show lethal virulence, although it did infect the muscles. On the other hand, when inoculated intranasally, all mutants grew in the lungs and killed immunosuppressed mice. Compared with PAO1, however, the pyoverdin-deficient mutant and the double mutant grew poorly in the lungs, and the latter was significantly attenuated for virulence. Irrespective of the inoculation route, the pyoverdin-deficient and doubly deficient mutants detected in the blood were significantly less numerous than PAO1. Additionally, in vitro examination demonstrated that the growth of the double mutant was extremely reduced under a free-iron-restricted condition with apotransferrin but that the growth reduction was completely canceled by supplementation with hemoglobin as a heme source. These results suggest that both pyoverdin and pyochelin are required for efficient bacterial growth and full expression of virulence in P. aeruginosa infection, although pyoverdin may be comparatively more important for bacterial growth and dissemination. However, the siderophores were not always required for infection. It is possible that non-siderophore-mediated iron acquisition, such as via heme uptake, might also play an important role in P. aeruginosa infections.

ERP development in the rat in the course of learning two-tone discrimination task.

To clarify some neurophysiological aspects of learning, we investigated the relationship between the course of learning and development of ERP and investigated developmental processes of ERPs. Nine male Sprague-Dawley rats were trained for a two-tone discrimination task and rat P3 and N1 component were longitudinally recorded. Both rat P3 and N1 gradually increased with learning only for target tones. An improvement in the proportion of correct responses preceded the increase in ERPs, and the increase in P3 and N1 proceeded almost simultaneously. These findings suggest that multiple kinds of information processing were acquired with learning the two-tone discrimination task. ERP development could be utilized as an index of establishment of learning.

Constituents in watercress: inhibitors of histamine release from RBL-2H3 cells induced by antigen stimulation.

Histamine release inhibitors in watercress (Nasturtium officinale) were isolated using a monitoring system with antigen-stimulated RBL-2H3 cells. Of the 15 compounds isolated, flavonols and megastigmanes significantly inhibited histamine release. Two flavonols, 3-O-sophorosides of rhamnetin and rhamnazin, were new compounds. To investigate the inhibitory mechanism, the effects of rhamnetin, rhamnetin 3-O-sophoroside and an isolated megastigmane glucoside on the increase in the intracellular free calcium concentration were examined at a concentration providing 60% inhibition of histamine release. The results suggest that these compounds did not affect the calcium influx at that concentration. The structure-activity relationships of the megastigmanes on histamine release were also investigated.

[Usefulness of continuous venous daily chemotherapy of 5-fluorouracil and low-dose cisplatin for patients undergoing noncurative surgery].

We examined the usefulness of continuous venous daily chemotherapy of 5-FU and low/I dose CDDP for patients undergoing noncurative surgery. Twelve patients with carcinoma (1 esophageal, 6 gastric, 4 colorectal carcinoma cases and 1 carcinoma case of unknown origin) underwent resection of primary lesion and postoperative adjuvant chemotherapy. The schedule for the chemotherapy was as follows: 2-7 courses of 24-hours continuous venous infusion of CDDP (5mg/day on day 1, 2, 3, 4, 5) and 5-FU (250 mg/day on day 1, 2, 3, 4, 5 or 1, 3, 5). Several patients received a bolus infusion of Epirubicin (10 mg/1 week or 30 mg/2 week). Mean total volumes of CDDP and 5-FU were 120 mg and 5.38 g, respectively. Nine cases could be evaluated. Results of the chemotherapy were 1 CR, 1 PR, 3 NC and 4 PD, and the overall response rate was 22%. Side effects were found in 6 cases, but were not so severe. One-year survival rate was 47%, and 50% survival time was 9 months. The prognosis of the patients seemed to be relatively good. These results suggest that continuous chemotherapy of 5-FU and low dose CDDP may be useful for patients undergoing noncurative surgery.

Extrinsic and intrinsic connections of the cat's lateral suprasylvian visual area.

The lateral suprasylvian visual area (LS) is known to have numerous interconnections with visual cortical areas as well as with subcortical structures implicated in visually-guided behaviors. In contrast, little data is available regarding connections within the LS itself. In order to obtain information about intra-areal connections and to re-investigate LS connectivity with various cortical and subcortical areas, the traces (biocytin or WGA-HRP) was injected into various loci along the medial and lateral banks of the LS. The anterograde tracer, biocytin injections into both medial and lateral bank produced label contained within the respective bank that extended rostrally and caudally from the infection site. In addition, following medical bank injections, considerable label was distributed throughout the fundus and, to a lesser extent, in the lateral bank. In contrast, no label could be detected in the medial bank after lateral bank injections, and, although label was observed in the fundus, it was restricted to the most lateral aspects. Moderate labeling could be observed in the medial bank following the tracer injection into the most rostral aspect of the lateral bank. It is likely that input derived from various visual cortical areas which project to the medial bank of the LS has access to this intra-areal circuitry. This may provide a route by which visual cortical information can be relayed to other cortical and subcortical structures involved in visually-guided behaviors such as the anterior ectosylvian visual cortex, striatum, and the deep layers of the superior colliculus, despite the fact that these structures themselves do not receive substantial direct projections from the visual cortical areas that are associated with the medial bank. Examination of the laminar location of the cells-of-origin of striate and extrastriate projections to LS using retrograde trace, WGA-HRP, revealed that the supragranular laminae of areas 17, 18 and 19 were the source of LS afferents whereas afferents from the other cortical areas (e.g., 20a, 20b, 21a, 21b, 7 and anterior ectosylvian visual area) were from both supra- and infragranular laminae. In addition, all LS subregions received intra-areal afferent projections from all LS cortical laminae. Thus, although rather clear hierarchical relationship between LS and visual cortical areas appears to exist, the interconnections among LS subregions provide no clear evidence of simple hierarchical relationships between regions LS or may have feed-forward and feed-back pathways.

Prophylactic portal infusion chemotherapy as adjuvant therapy for the prevention of metachronous liver metastasis in colorectal cancer.

The DNA ploidy and DNA indices (DI) of 414 patients with colorectal cancer were analyzed, and the incidence of patients with metachronous liver metastasis was found to be significantly higher in those with aneuploid tumors and a DI above 1.5 than in those with aneuploid tumors and a DI below 1.4, or in those with diploid tumors and a DI equal to 1.0. Next, to confirm the effectiveness of administering prophylactic portal infusion chemotherapy (PPIC) as adjuvant therapy for the prevention of metachronous liver metastasis in colorectal cancer, a randomized controlled trial of PPIC was performed on 110 consecutive patients with primary colorectal cancer who had undergone curative resection. Although the incidence of patients with metachronous liver metastasis in the two study groups was not significantly different at 3.3% in the PPIC group and 10.3% in the control group, the incidence in the patients with aneuploidy and a DI above 1.5 was significantly lower in the PPIC group than in the control group. These findings suggest that colorectal cancer with aneuploidy and a DI above 1.5 may have a strong tendency to metastasize to the liver, and that prophylactic portal infusion chemotherapy may be effective for preventing metachronous liver metastasis in such patients.

Hypothalamic GnRH pulse generator activity during the estradiol-induced LH surge in ovariectomized goats.

Electrophysiological manifestation of the hypothalamic GnRH pulse generator activity was examined during the LH surge induced by estradiol in ovariectomized goats. The characteristic increases in the frequency of multiple unit activity (MUA volley) associated with the pulsatile secretion of LH were recorded using electrodes implanted bilaterally in the medial basal hypothalamus. Estradiol was infused for 16 h at the rate of 3 micrograms/h, to induce an LH surge 10.0-11.5 h later. Regular recurrence of MUA volley was observed throughout the experimental period including the LH surge, but the interval between the MUA volleys became longer (p < 0.01) after the onset of the LH surge as compared with the pretreatment control period (32.9 +/- 2.1 vs. 60.0 +/- 5.1 min). These results suggest that an increased frequency of LH pulses is not a prerequisite for the LH surge in ovariectomized goats given estradiol, and imply that the positive feedback effects of estradiol on LH secretion appear to be exerted through a neuronal mechanism that is intrinsically different from the GnRH pulse generator.

Chronic recording of electrophysiological manifestation of the hypothalamic gonadotropin-releasing hormone pulse generator activity in the goat.

A characteristic increase in multiple-unit activity (MUA volley) synchronized with the pulsatile secretion of luteinizing hormone (LH) by the pituitary gland was recorded from electrodes chronically implanted in the medial basal hypothalamus of ovariectomized goats. The MUA volley always preceded the LH pulse, and the temporal correlation was consistent even when the pulse frequency was altered with an anesthetic or exogenous steroids. The electrophysiological manifestation of the hypothalamic 'pulse generator' governing the secretion of gonadotropin-releasing hormone was thus demonstrated in the ruminant.