Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer's disease therapy.

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

Lower values of handgrip strength and adductor pollicis muscle thickness are associated with hepatic encephalopathy manifestations in cirrhotic patients.

Hepatic encephalopathy (HE) is a late complication of liver cirrhosis and is clearly associated with poor outcomes. Chronic liver insufficiency leads to progressive muscle wasting, impairing ammonia metabolism and thus increasing the risk for HE. Given the association between lean mass and adductor pollicis muscle thickness (APMT), it has been used to predict outcome and complications in many conditions, but not yet in cirrhotic patients. Therefore, this article aimed to study the association between HE manifestations and measures related to muscle mass and strength. This cross-sectional study included 54 cirrhotic outpatients with HE varying from subclinical to grade II according to the West-Haven criteria, who were submitted to neuropsychometric tests, electroencephalogram, brain Single Photon Emission Computed Tomography (SPECT), anthropometric measurements, handgrip strength (HGS) and dual energy X-ray absorptiometry exam (DXA). Multiple logistic regression analysis was performed to investigate the association between body composition measures and HE grade. Analysis of the area under the receiver operator characteristic (AUROC) curve revealed the values related to neurological manifestations (HE grades I and II). Reductions in APMT and HGS were associated with higher HE grades, suggesting a big impact caused by the loss of muscle mass and function on HE severity. The link between HE manifestations and anthropometric measures, namely APMT and HGS, point to a significant relation concerning skeletal muscles and the neurological impairment in this population.

The vanadium (IV) compound rescues septo-hippocampal cholinergic neurons from neurodegeneration in olfactory bulbectomized mice.

The bilateral olfactory bulbectomy (OBX) mouse exhibits neurodegeneration of cholinergic neurons in the medial septum with concomitant cognitive deficits. Consistent with our previous observations, choline acetyltransferase (ChAT) protein levels in the medial septum decreased by 43.5% 2 weeks after OBX without changes in glutamic acid decarboxylase-65 (GAD65) levels. Interestingly, levels of the vesicular acetylcholine transporter (VAChT), which is localized at cholinergic neuron terminals, decreased both in hippocampal CA1 and CA3 regions following OBX. Confocal microscopy showed that VAChT expression was more severely reduced in CA3 14 days after OBX compared with CA1. Intriguingly, chronic treatment with a vanadium (IV) compound, VO(OPT) [bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV)] (0.5-1 mg as vanadium (V)/kg/day, i.p.), significantly rescued cholinergic neurons in the medial septum in a dose-dependent manner. VO(OPT) treatment also prevented decreased VAChT immunoreactivity both in CA1 and CA3 regions in the hippocampus. Consistent with these findings, an impaired hippocampal long-term potentiation (LTP) and memory deficits seen in OBX mice were significantly prevented by VO(OPT) treatment. Taken together, OBX induces neurodegeneration of septo-hippocampal cholinergic neurons and impairment of memory-related behaviors. The neuroprotective effect of VO(OPT) could lead to novel therapeutic strategies to ameliorate cognitive deficits associated with cholinergic neuron degeneration in Alzheimer's disease and other neurodegenerative disorders.

Activation of phosphatidylinositol 3-kinase/protein kinase B pathway by a vanadyl compound mediates its neuroprotective effect in mouse brain ischemia.

We previously reported that orthovanadate composed of vanadate (V(5+)) activates phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling through inhibition of protein tyrosine phosphatases, thereby eliciting neuroprotection in brain ischemia/reperfusion injury. However, therapeutic doses of orthovanadate are associated with diarrhea due to inhibition of ATPase. By contrast, vanadyl (V(4+)) organic compounds show low cytotoxicity. Since both vanadate and vanadyl inhibit protein tyrosine phosphatases, we tested whether bis(1-oxy-2-pyridinethiolato)oxovanadium(IV) [VO(OPT)] in a vanadyl form elicits a neuroprotection in brain ischemia. In a mouse transient middle cerebral artery occlusion (MCAO) model, pre- and post-treatments with VO(OPT) significantly reduced infarct volume in a dose-dependent manner. Like orthovanadate, activation of the PI3K/Akt pathway mediated neuroprotective action. VO(OPT) treatment inhibited reduced Akt phosphorylation at Ser-473 following brain ischemia and restored decreased phosphorylation of forkhead box class O (FOXO) family members such as FKHR, FKHRL1, and AFX. Consistent with inhibition of FOXO dephosphorylation, VO(OPT) treatment blocked elevated expression of Fas-ligand, Bim and active caspase-3 24 h after ischemia/reperfusion. Taken together, a vanadyl compound, VO(OPT) elicits neuroprotective effects on brain ischemia/reperfusion injury without apparent side effects.

Effects of dietary retinyl palmitate or 13-cis-retinoic acid on the promotion of tumors in mouse skin.

The present study was designed to determine the effects of dietary 13-cis-retinoic acid and retinyl palmitate on mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Female CD-1 mice were initiated with 150 nmol of 7,12-dimethylbenz(a)anthracene and promoted twice weekly with 8 nmol of TPA. Diets supplemented with retinyl palmitate to yield 60,000 or 200,000 IU or 700,000 for 5 wk followed by 350,000 IU per kg of diet (700,000/350,000) fed to mice during tumor promotion resulted in 9%, 37%, and 65% inhibition of the papilloma yield, respectively, at 21 wk of promotion. Although topical applications of 13-cis-retinoic acid have been almost as effective as retinoic acid in preventing the appearance of mouse skin tumors, dietary 13-cis-retinoic acid at 200,000 or 700,000 IU per kg of diet resulted in no reduction in papilloma yield but did result in a dose-dependent decrease in the tumor burden (weight of tumors per mouse). Therefore, dietary retinyl palmitate yielded a dose-dependent inhibition of the number and weight of tumors promoted by TPA, whereas dietary 13-cis-retinoic acid resulted in a decrease in weight but not in number of tumors promoted by TPA.

In vitro generation of tumoricidal properties in human alveolar macrophages following interaction with endotoxin.

Human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy nonsmoking donors exhibited primarily low levels of cytolytic activity against allogeneic tumor target cells. These AM acquired enhanced capacity to kill tumor cells following a 24-hr incubation in vitro with endotoxin [lipopolysaccharide (LPS)]. Maximal tumoricidal activity of LPS-activated AM as measured by lysis of tumor target cells was obtained after incubation with tumor cells for 72 hr. LPS-activated AM lysed allogeneic tumor cell lines of different origins but did not affect normal, nonneoplastic cells. We conclude that LPS induces human AM to become tumoricidal. This method should be useful in studies on therapeutic agents enhancing AM-mediated cytotoxicity in situ.

Prognostic significance of argyrophilic nucleolar organizer regions in esophageal carcinoma.

The argyrophilic nucleolar organizer region (AgNOR) of 100 cancer cells from biopsy specimens of esophageal squamous cell carcinomas in 98 surgically treated cases was examined, using a silver colloid staining technique on biopsy specimens. The number of AgNOR per nucleus (AgNOR number) was higher in the more advanced groups with regard to the length of the tumor (P less than 0.01), the depth of penetration (P less than 0.05), and lymph node metastasis (P less than 0.01). The survival of the patients with a high AgNOR number (greater than or equal to 6) was significantly poorer than those with either a medium range AgNOR number (4 less than or equal to-less than 6) (P less than 0.05) or a low AgNOR number (less than 4) (P less than 0.01). In the multivariate analysis including conventional clinicopathological factors, the AgNOR number was found to be one of the independent and significant variables (P less than 0.01). Because the AgNOR method is simple and can be applied to paraffin-embedded sections, the AgNOR number may provide potential benefit in the pretherapeutic assessment of malignant potentiality in esophageal carcinoma.

Effects of selenium in vitro on human T-lymphocyte functions and K-562 tumor cell growth.

In vitro E-rosette formation, lymphocyte mitogenesis, and natural killer (NK) cell activity of human blood lymphocytes were strongly inhibited by high concentrations (10(-4) M) of sodium selenite, sodium selenate, and selenium dioxide. Lower concentrations (10(-5) or 10(-7) M) also inhibited E-rosette formation and natural killer cell activity against K-562 tumor cells. Lymphocyte transformation induced by concanavalin A (con A) or pokeweed mitogen (PWM) was also inhibited by all selenium compounds tested, but only at the highest concentrations (10(-5) and 10(-4) M). There was depression of the total number of viable lymphocytes following incubation with selenium dioxide only at a high concentration (10(-4) M). Interferon production was enhanced at lower levels (10(-9) to 10(-6)M) of selenium dioxide while a higher concentration (10(-5) and 10(-4)M) appeared to inhibit its production. The mechanism of inhibition by selenium compounds (10(-4) M) is due, in part, to the decrease of viable lymphocytes. It is unclear how other and lower concentrations (10(-7) or 10(-9) M) of selenium compounds inhibit E-rosette formation, NK activity, or K-562 tumor cell growth.

Decreased mitogen response of splenic lymphocytes in obese Zucker rats is associated with the decreased expression of glucose transporter 1 (GLUT-1).

We reported previously that obesity is a risk factor for deteriorating cellular immune functions in aging. However, the mechanism by which obesity decreases cellular immunity remains to be elucidated. To determine the mechanism of the decrease in cellular immunity with obesity, lean (Fa/?) and obese (fa/fa) 12-mo-old Zucker rats were used. The mitogen response of splenic lymphocytes in obese Zucker rats was significantly lower than that of lean Zucker rats, which was not restored by in vitro treatment with indomethacin (10 micromol/L), an inhibitor of prostaglandin E2 (PGE2). In addition, PGE2 production by splenic lymphocytes was not greater in obese than in lean Zucker rats. Glucose consumption by splenic lymphocytes after in vitro incubation with concanavalin A (conA) for 48 h was also significantly lower in obese Zucker rats. Expression of glucose transporter 1 (GLUT-1), analyzed by Western blot analysis, was lower in splenic lymphocytes of obese than in lean Zucker rats. However, the expression of the conA receptor in splenic lymphocytes, analyzed by flow cytometry with fluorescein isothiocyanate-conjugated conA, was not significantly different between lean and obese Zucker rats. In conclusion, the decreased mitogen response of splenic lymphocytes in obese Zucker rats may be due in part to the decreased uptake of glucose as the main energy source for lymphocytes at the stage of proliferation and may be associated with the decreased expression of GLUT-1.

Effects of dietary retinyl palmitate and selenium on tumoricidal capacity of macrophages in mice undergoing tumor promotion.

Dietary retinyl palmitate was administered for 22-30 weeks in CD-1 mice which had been initiated with 0.15 mumol of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted with 8 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly thereafter. This treatment resulted in a dose response in both the tumoricidal capacity of a selected number of isolated peritoneal macrophages (PM) and in skin tumor prevention. At 350 I.U./g of diet, retinyl palmitate (RP) also resulted in a 3-fold increase in the number of DM. RP significantly increased the total capacity of macrophage host defenses by increasing the number and individual capacity for cytotoxicity. Selenium (Se), at 2 parts/million in the drinking water, did not enhance PM tumoricidal capacity, although it did result in 60% reduction of mouse tumor burden.

DNA ploidy is a major prognostic factor in advanced gastric carcinoma--univariate and multivariate analysis.

DNA ploidy was determined by cytofluorometric analysis of paraffin-embedded malignant tissue from 96 Japanese patients in whom gastric carcinoma had invaded the serosa. Aneuploidy was found in gastric carcinoma tissue from 63 patients (66%). The postoperative 5-year survival rate of patients with aneuploid malignancy was significantly lower (13%) than those with diploid malignancy (36%) (p less than 0.05). A multivariate analysis of various clinical and pathologic factors showed that tumor size, lymph node metastasis, vascular invasion, and DNA ploidy were significant and independent factors, which correlated with prognosis.

Pertinent risk factors and gastric carcinoma with synchronous peritoneal dissemination or liver metastasis.

From 1965 to 1985, of 1108 patients with advanced gastric cancer who underwent gastric resection in our department, 216 patients (19.5%) had synchronous peritoneal dissemination (PD) or liver metastasis (LM). The 1-year survival rate was 22.5% for PD, 14.7% for LM, and 4.8% for PD plus LM (p less than 0.01). With PD, patients were younger, tumor was larger, Borrmann type 3 and 4 and undifferentiated lesions were more frequent, and the incidence of serosal invasion was higher. Histologic findings showed a pattern of infiltrative growth. With LM, patients were more likely to be male, Borrmann type 2 and 3 and differentiated lesions were frequent, and the antral region was often involved. The degree of serosal invasion was less than with PD. The pattern of growth was the expansive type and vascular involvement was prominent. Rate of lymph node metastasis was high in both groups. The route of metastasis was partly linked to features of the primary lesion. Multivariate analysis showed that independent risk factors involved in the occurrence of each metastasis are serosal invasion, lymph node metastasis, and undifferentiated tissue type for PD, and lymph node metastasis and vascular involvement for LM. When designing treatment in an attempt to suppress a recurrence, even after curative resection, all of these risk factors must be kept in mind.

The prognostic significance of proliferating cell nuclear antigen in clinical gastric cancer.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is an intranuclear protein that is closely linked to the cell cycle. This antigen can be detected in formalin-fixed specimens. We studied the expression of PCNA in primary gastric cancer to identify its significance as a prognostic factor. METHODS: The avidin-biotin-peroxidase complex method was used for PCNA staining in sections from 138 patients with primary gastric cancer. All sections were formalin fixed and paraffin embedded. Two different sections were examined in each case. RESULTS: The PCNA labeling index (PCNA-positive cells/1000 cells x 100) varied from 7.0% to 59.7%. There were significant differences in tumor size, morphologic type, depth of tumor invasion, lymphatic permeation, vascular permeation, and lymph node metastasis between the high (> or = 33.4) and low (< 33.4) PCNA labeling index groups. The 5-year survival rates of the high and low PCNA labeling index groups were 5.8% and 66.2%, respectively, a significant difference (p < 0.001). Multivariate analysis showed that the PCNA labeling index was an independent prognostic factor for gastric cancer. CONCLUSIONS: Because PCNA immunostaining can be done in routine formalin-fixed paraffin-embedded sections, this could be a powerful tool for providing information about the prognosis of patients with gastric cancer.

Exercise training restores decreased cellular immune functions in obese Zucker rats.

This study investigated whether exercise training had a beneficial effect on the decreased mitogen response and improved a decreased expression of glucose transporter 1 (GLUT-1) in splenocytes from obese Zucker rats. Experimental groups were lean and sedentary and exercise-trained obese Zucker rats. Exercise training, running on a motor-driven treadmill for 5 days/wk for 40 wk, did not induce a significant decrease in body weight in obese Zucker rats. The plasma insulin concentration, showing a significant increase compared with lean Zucker rats, was unaffected by exercise training. However, the plasma triglyceride concentration in obese Zucker rats was significantly depressed by exercise training, whereas it was still higher than that in lean Zucker rats. In addition, natural killer cell activity and concanavalin A-induced mitogenesis of splenic lymphocytes of obese Zucker rats were significantly restored. In these splenic lymphocytes, glucose uptake was significantly lower compared with that in lean Zucker rats, which was also improved by exercise training. Although the expression of GLUT-1, the major glucose transporter in immune cells, was depressed in splenic lymphocytes of obese Zucker rats, exercise training induced a significant improvement. These results suggest that exercise training has a beneficial effect on the decreased cellular immune functions in obese Zucker rats, which is associated, in part, with the improvement in GLUT-1 expression.

Identification of casoxin C, an ileum-contracting peptide derived from bovine kappa-casein, as an agonist for C3a receptors.

Casoxin C (Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg) is a bioactive peptide that was isolated from a tryptic digest of bovine kappa-casein as an anti-opioid peptide in longitudinal strips of guinea pig ileum. Casoxin C also evokes contraction of the ileal strips, and we found that this process was biphasic with rapid and slow components. The contractile profile was very similar to that of human complement C3a(70-77), which is the COOH-terminal octapeptide of C3a and has, although less potent, qualitatively the same biological activities as C3a. Casoxin C also has homology with C3a(70-77). The rapid contraction was mediated by histamine release and the slow contraction was mediated by a prostaglandin E2-like substance, judging from the effects of various pharmacological inhibitors and antagonists on the ileal contraction. Casoxin C had affinity for C3a receptors (IC50 = 40 microM) in the radioreceptor assay. In addition, casoxin C showed phagocyte-stimulating activities. Casoxin C is therefore the first milk-derived peptide identified, that acts through complement C3a receptors.

Studies on the ileum-contracting mechanisms and identification as a complement C3a receptor agonist of oryzatensin, a bioactive peptide derived from rice albumin.

Oryzatensin (Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg) is an ileum-contracting and immunostimulating peptide derived from rice albumin. The mechanisms for the ileal contraction that it induces, consisting of rapid and slow components, were examined. The rapid contraction was mediated by histamine release and the slow contraction by a prostaglandin E2-like substance, judging from the effects of various pharmacological inhibitors and antagonists on ileal contraction and titration of histamine release. The contractile profile was very similar to that of human complement C3a(70-77), which is the COOH-terminal octapeptide of C3a and has, although less potent, qualitatively the same biological activities as C3a. Oryzatensin showed homology with C3a(70-77) and affinity for C3a receptors (IC50 = 44 microM) by radioreceptor assay. This is the first report of a food-derived bioactive peptide acting through complement C3a receptors.

Multivariate analysis of postoperative complications after esophageal resection.

To determine the contributing factors for eight postoperative complications after esophagectomy through a right thoracoabdominal approach, a multivariate analysis was carried out on preoperative and intraoperative variables in 141 patients with thoracic esophageal cancer. Although postoperative complications occurred in 125 patients, only 7 died of such complications. The multivariate analysis indicated that the retrosternal route was a significant factor predisposing to postoperative atelectasis. Age, preoperative arterial oxygen tension, and volume transfused were significant factors predisposing to postoperative hypoxemia, whereas age, routes other than the intrathoracic route, and volume transfused were significant factors predisposing to prolonged respiratory support. In addition, preoperative total serum bilirubin level and volume transfused were significant factors predisposing to postoperative hyperbilirubinemia; preoperative serum creatinine level was a significant contributing factor for postoperative renal insufficiency; and sex, antesternal route, and substituted colon were significant contributing factors for anastomotic leakage. There were no significant factors predisposing to postoperative pneumonia and liver dysfunction. These significant factors should be taken into consideration not only during perioperative management but also when choosing the operative procedures and extending the surgical indication for esophagectomy through a right thoracoabdominal approach.

Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity.

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.

Central administration of a nitric oxide synthase inhibitor causes pressor responses via the sympathetic nervous system and the renin-angiotensin system in Wistar rats.

The central roles of nitric oxide (NO) in regulations of the blood pressure and heart rate were examined in anesthetized rats. Intracerebroventricular (i.c.v.) injection of Nomega-nitro-L-arginine methyl ester (L-NAME) caused dose-dependent increase in the blood pressure and heart rate. The pressor response of the blood pressure to L-NAME (2 micromol, i.c.v.) was reduced by L-arginine (5 micromol, i.c.v). Pretreatment with a ganglionic blocker, pentolinium (10 mg/kg, i.v.), significantly inhibited both pressor responses induced by L-NAME (2 micromol, i.c.v). The later pressor response of the blood pressure to L-NAME was also inhibited by the angiotensin II AT-1 blocker losartan (10 mg/kg, i.v). These results suggest that the response of the blood pressure to L-NAME is mediated by both the sympathetic nervous system and the renin-angiotensin system.

Risk factors which predict pattern of recurrence after curative surgery for patients with advanced gastric cancer.

The objective of the study was to define risk factors for peritoneal dissemination and haematogenous metastasis after curative resection of patients with an advanced gastric cancer. In retrospective analyses of 405 patients, 168 died of a tumour recurrence. Patients who died of gastric cancer were more likely to have large, invasive tumours which had spread throughout the stomach, metastasized to lymph nodes, and vessel invasion by gastric cancerous cells (P < 0.01 or P < 0.05). Of the 168 deaths, 60 (35.7%) were secondary to haematogenous recurrence, 53 (31.5%) were related to peritoneal dissemination, and 19 (11.3%) were related to a local recurrence. To determine the independent risk factors related to peritoneal dissemination and haematogenous metastasis, multivariate analyses using a stepwise logistic model suggested that serosal invasion (P < 0.01, relative risk = 2.57) and Borrmann type 4 (P < 0.01, relative risk = 1.95) were the greatest risk factors for peritoneal dissemination. The presence of lymph node metastasis (P < 0.01, relative risk = 2.62) and presence of vessel invasion by cancerous cells (P < 0.05, relative risk = 1.59) were the greatest risk factors for a haematogenous metastasis.