Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

OBJECTIVE: In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. METHODS: Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. RESULTS: High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. CONCLUSIONS: ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma.

A new operative technique for the resection of gastric tube cancer by means of lifting the anterior chest wall and videoscope-assisted surgery.

The prolonged survival of patients receiving surgery for esophageal cancer has led to an increased incidence of adenocarcinoma arising in the gastric tube used for reconstruction (gastric tube cancer). In patients with advanced gastric tube cancer, resection of the gastric tube should be considered, but currently available procedures are very invasive. In patients undergoing curative surgery for gastric tube cancer that has developed after reconstruction through the retrosternal route, the gastric tube is usually resected through a median sternotomy, followed by reconstruction with the colon. However, postoperative complications often occur and treatment outcomes remain poor. We developed a new surgical technique for gastric tube resection without performing a sternotomy in patients with gastric tube cancer who had previously undergone reconstruction through the retrosternal route. Our technique was used to treat two patients. Two Kirschner wires were passed subcutaneously through the anterior chest; the chest was lifted to extend the retrosternal space and secure an adequate surgical field. The stomach was separated from the surrounding tissue under videoscopic guidance. Total resection of the gastric tube was done. The retrosternal space was used to lift the jejunum. Roux-en-Y reconstruction was performed. Neither patient had suture line failure or surgical site infection. Their recovery was uneventful. Our surgical technique has several potential advantages including (i) reduced surgical stress; (ii) the ability to use the retrosternal space for reconstruction after gastric tube resection; and (iii) a reduced risk of serious infections such as osteomyelitis in patients with suture line failure. Our findings require confirmation by additional studies.

Chemosensitivity of patients with recurrent esophageal cancer receiving perioperative chemotherapy.

Perioperative chemotherapy (CT) and chemoradiotherapy are widely used for advanced esophageal cancer. We evaluated the chemosensitivity of patients displaying recurrent esophageal cancer after esophagectomy with perioperative CT. From the database at National Cancer Center Hospital in Tokyo, we extracted recurrent esophageal cancer cases after perioperative CT and evaluated the effectiveness of the first CT against the recurrent disease according to the duration between termination of the original perioperative CT and recurrence with treatment-free intervals (TFIs) 6 months. Systemic CT for their recurrent disease was performed for 30 esophageal cancer patients after perioperative CT. All patients received 5-fluorouracil and cisplatin as perioperative CT, with relapses occurring at TFIs 6 months in 19 patients (all received platinum-containing regimens). The response rate of patients experiencing a recurrence at TFIs 6 months was 0 and 37% (P = 0.029), the median progression-free survival was 2.8 and 4.8 months (log-rank P = 0.001) and the median overall survival was 6.1 and 10.2 months (log-rank P = 0.012), respectively. Recurrence at the TFI

Transition of Akabane virus genogroups and its association with changes in the nature of disease in Japan.

Akabane virus (AKAV) is teratogenic to the foetus of domestic ruminants and causes a significant reproduction loss in cattle in Japan. In several past epizootics in cattle, AKAV was also associated with post-natal encephalomyelitis, mainly in calves and young stock. Previously analysed AKAV isolates in East Asia form two major clusters, genogroups I and II, with isolates involved in encephalomyelitis belonging mainly to the former. Between 2007 and 2013, AKAV epizootics were regularly observed in Japan during the summer/autumn season, and abnormal deliveries and post-natal encephalomyelitis caused by the virus in cattle were reported. During this period, 30 AKAV isolates were obtained from diseased and sentinel cattle, a piglet and Culicoides biting midges throughout Japan and were subjected to genetic comparison and phylogenetic analysis with previous isolates. In 2007, 2011 and 2013, AKAV belonging to genogroup I was identified in the central nervous systems of calves showing neurological disorders. Notably, a total of 165 cases of bovine encephalomyelitis were reported in 2011 and the isolated viruses from affected animals shared high genetic identities with a South Korean isolate that was associated with a large outbreak in 2010, suggesting some epidemiological linkage between these epizootics. Epizootics of genogroup II were observed in 2008 and 2010, but bovine post-natal encephalomyelitis cases rarely occurred. Our findings suggest that the frequent incursion of genogroup I isolates has increased the frequency of post-natal encephalomyelitis cases in Japan in recent years. Infection by genogroup I virus was also identified in piglets with neurological disorders or congenital malformations in 2011 and 2013. The aetiological role of AKAV in pigs should be elucidated in the future.

Inhibition of tumor cell invasion through matrigel by a peptide derived from the domain II region in urinary trypsin inhibition.

Urinary trypsin inhibitor (UTI) has a multipotent inhibitory effect on proteases such as trypsin, chymotrypsin, plasmin, human leukocyte elastase, or hyaluronidase. UTI can bind easily to its receptors on various types of tumor cells (human ovarian cancer HOC-I cells, human choriocarcinoma SMT-cc1 cells, and murine Lewis lung carcinoma 3LL cells). Our results show that the UTI receptors of some tumor cells have a possible role in modulating plasmin activity on the cell surface and prevention of tumor cell invasion and metastasis (H. Kobayashi et al., J. Biol. Chem., 269; 20642-20647, 1994). UTI interacts with tumor cells as a negative modulator of the invasive cells. We investigated whether this effect may be mediated by UTI binding to the cell surface receptors. In addition, the role of peptide sequences from each UTI domain and their interaction with tumor cells were investigated. UTI derivatized with biotin or FITC was taken up by tumor cells in a dose-dependent manner. This cell association was inhibited with a monoclonal antibody D1, which specifically recognizes NH2 terminus (domain I) of UTI. The binding was inhibited by fluid phase UTI, but not HI-8, COOH terminus (domain II) of UTI, suggesting that UTI binds to cells through a site in the UTI domain I. Furthermore, we found that UTI, HI-8 and a number of peptides containing Arg-Gly-Pro-Cys-Arg-Ala-Phe-Ile promoted the inhibition of tumor cell invasion. This site corresponds to the plasmin-inhibiting domain within HI-8. The possibility that UTI binding to tumor cells might be involved in the prevention of tumor cell invasion in vitro was excluded since HI-8, lacking domain I, promotes the inhibition of tumor cell invasion with essentially the same affinity as UTI. All these data allow us to conclude that inhibition of tumor cell invasion is mediated by domain II, which possesses anti-plasmin activity.

A phase I study of binimetinib (MEK162) in Japanese patients with advanced solid tumors.

PURPOSE: Binimetinib is a potent, selective MEK1/2 inhibitor with demonstrated efficacy against BRAF- and RAS-mutant tumors. Retinal adverse events associated with MEK inhibitors have been reported in some cases. The aim of this study was to assess single-agent binimetinib, with detailed ophthalmologic monitoring, in Japanese patients with advanced solid tumors. METHODS: This was an open-label phase I dose-escalation and dose-expansion study (NCT01469130). Adult patients with histologically confirmed, evaluable, advanced solid tumors were enrolled and treated with binimetinib 30 or 45 mg twice daily (BID). The primary objective was to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) of single-agent binimetinib in Japanese patients. RESULTS: Twenty-one patients were enrolled; 3 and 8 patients had documented BRAF and KRAS mutations, respectively. Two of 6 patients (33 %) receiving binimetinib 45 mg BID in dose-escalation experienced recurrent grade 2 retinal adverse events (AEs) which were reversible, and this dose was declared the MTD and RP2D. All patients experienced ≥1 AE suspected to be treatment related; the most common (>50 %) were blood creatine phosphokinase increase (76 %), retinal detachment and aspartate aminotransferase increase (62 % each), and diarrhea (52 %). There were no complete or partial responses; 14 patients (67 %) had stable disease, which lasted >180 days in 5 patients. Expression of phospho-ERK decreased in the skin following binimetinib treatment at both dose levels, indicating target inhibition. CONCLUSIONS: Binimetinib demonstrated efficacy and acceptable safety in Japanese patients with solid tumors, supporting the 45 mg BID dose of binimetinib as the RP2D.

Suppression of urokinase-type plasminogen activator expression from human ovarian cancer cells by urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, efficiently inhibits tumor cell invasion and metastasis. We examined the effect of UTI on urokinase-type plasminogen activator (uPA) expression in ovarian cancer cell lines, HOC-I and HRA. By Northern blot, Western blot, ELISA, and zymographic analyses, we demonstrated that UTI inhibited the expression of uPA mRNA and protein in these cells in a time- and dose-dependent manner, independent of whether induction was triggered by phorbol ester. Monoclonal antibody 4G12, which inhibits UTI binding to the cells, produced a dose-dependent abrogation in UTI-mediated down-regulation of uPA expression. These data suggest that UTI significantly down-regulates tumor cell uPA mRNA expression and protein secretion, and that UTI binding to the cells is necessary to exert the UTI's action.

Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins.

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC(50) of inhibiting binding of (125)I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC(50) for inhibiting binding of (125)I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.

Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine.

We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.

Identification and characterization of the cell-associated binding protein for urinary trypsin inhibitor.

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20,642-20,647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH2-terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan-hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.

Identification of link protein during follicle development and cumulus cell cultures in rats.

Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid production and accumulation of proteins of the inter-alpha-trypsin inhibitor family in the gonadotropin-stimulated cumulus cells. Link protein, a glycoprotein found in cartilage, interacts specifically with hyaluronic acid and stabilizes the binding of proteoglycan monomers to hyaluronic acid to form aggregates. The aim of this study was to investigate the expression of immunoreactive link protein during follicle development in rats and in cumulus cells in culture by immunohistochemistry and Western blot as well as by specific enzyme-linked immunosorbent assay. Immunohistochemical analysis revealed that the extracellular matrix of cumulus cells that were morphologically at a stage of COC expansion were markedly stained for link protein, whereas granulosa cells from immature follicles were not stained. Cumulus cells deposited link protein into the extracellular matrix in an in vitro culture system. The staining intensity was negated by the treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid. We have identified a 42-kDa immunoreactive link protein in rat ovary during the preovulatory period and in COC extracts. Addition of FSH to the medium of cumulus cells in culture supplemented with 10% FBS and oocyte-conditioned medium resulted in an increased rate of link protein synthesis. This work suggests that the cumulus cells synthesize the link protein that may stabilize the binding of inter-alpha-trypsin inhibitor or dermatan sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.

Right sylvian fissure subarachnoid hemorrhage has electrocardiographic consequences.

BACKGROUND AND PURPOSE: Abnormal ECG changes are frequently observed in patients with subarachnoid hemorrhage (SAH). Recently, evidence has been obtained that right insular cortex mediates sympathetic cardiovascular effects. We therefore assessed the laterality and location of SAH dominance in inducing cardiovascular changes as measured by ECG, blood pressure, and heart rate. METHODS: After exclusion of 11 SAH patients who died within 1 month after onset, we studied 118 consecutive patients. Data were obtained from records of blood pressure and pulse on admission. Abnormal ECG changes were determined from ECGs on admission and almost 1 month later. From brain CT scans performed immediately after admission, the amount of SAH in each of the 8 cisterns and fissures was measured semiquantitatively. RESULTS: Twenty-six patients had abnormal changes on admission ECG, while 92 patients did not. Systolic blood pressure, diastolic blood pressure, and the amounts of blood in the left ambient cistern, left suprasellar cistern, quadrigeminal cistern, right ambient cistern, right suprasellar cistern, right sylvian fissure, and the set of all cisterns were significantly greater in the group with ECG change than in the group without ECG change. Multivariate logistic regression analysis with stepwise method indicated that systolic blood pressure >160 mm Hg (P=0.0006) and the amounts of SAH in the quadrigeminal cistern (P=0.022) and right sylvian fissure (P=0.0019) were independently associated with abnormal ECG change. CONCLUSIONS: Cardiac consequences are possible in patients with massive right sylvian fissure SAH or when systolic blood pressure is >160 mm Hg.

Heterozygous knockout of the IRS-1 gene in mice enhances obesity-linked insulin resistance: a possible model for the development of type 2 diabetes.

Insulin receptor substrate 1 (IRS-1) gene polymorphisms have been identified in type 2 diabetic patients; however, it is unclear how such polymorphisms contribute to the development of diabetes. Here we introduced obesity in heterozygous IRS-1 knockout (IRS-1(+/-)) mice by gold-thioglucose (GTG) injection and studied the impact of reduced IRS-1 expression on obesity-linked insulin resistance. GTG injection resulted in approximately 30% weight gain in IRS-1(+/-) and wild type (WT) mice, compared with saline-injected controls. There was no difference in insulin sensitivity between lean IRS-1(+/-) and lean WT. Elevated fasting insulin levels but no change in fasting glucose were noted in obese IRS-1(+/-) and WT compared with the respective lean controls. Importantly, fasting insulin in obese IRS-1(+/-) was 1.5-fold higher (P<0.05) than in obese WT, and an insulin tolerance test showed a profound insulin resistance in obese IRS-1(+/-) compared with obese WT. The islets of obese IRS-1(+/-) were 1.4-fold larger than those of obese WT. The expression of insulin receptor and IRS-1 and IRS-2 was decreased in obese IRS-1(+/-), which could in part explain the profound insulin resistance in these mice. Our results suggest that IRS-1 is the suspected gene for type 2 diabetes and its polymorphisms could worsen insulin resistance in the presence of other additional factors, such as obesity.

Insulin down-regulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway.

Insulin receptor substrate (IRS)-1 and IRS-2 are the major substrates that mediate insulin action. Insulin itself regulates the expression of the IRS protein in the liver, but the underlying mechanisms of IRS-1 and IRS-2 regulation are not fully understood. Here we report that insulin suppressed the expression of both IRS-1 and IRS-2 proteins in Fao hepatoma cells. The decrease in IRS-1 protein occurred via proteasomal degradation without any change in IRS-1 mRNA, whereas the insulin-induced suppression of IRS-2 protein was associated with a parallel decrease in IRS-2 mRNA without changing IRS-2 mRNA half-life. The insulin-induced suppression of IRS-2 mRNA and protein was blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, but not by the MAP kinase-ERK kinase (MEK) inhibitor, PD098059. Inhibition of Akt by overexpression of dominant-negative Akt also caused complete attenuation of the insulin-induced decrease in IRS-2 protein and partial attenuation of its mRNA down-regulation. Some nuclear proteins bound to the insulin response element (IRE) sequence on the IRS-2 gene in an insulin-dependent manner in vitro, and the binding was also blocked by the PI 3-kinase inhibitor. Reporter gene assay showed that insulin suppressed the activity of both human and rat IRS-2 gene promoters through the IRE in a PI 3-kinase-dependent manner. Our results indicate that insulin regulates IRS-1 and IRS-2 through different mechanisms and that insulin represses IRS-2 gene expression via a PI 3-kinase/Akt pathway.

Expression of urokinase and urinary trypsin inhibitor in metastatic and non-metastatic murine Lewis lung carcinoma cells.

The murine Lewis lung carcinoma 3LL cells give rise to spontaneous and experimental lung metastasis in C57BL/6 mice. Tumor cells maintained by serial subcutaneous transplantation in mice retain their ability to form lung metastasis, while cells carried in vitro loose metastatic potential with time. In order to obtain the non-metastatic subline, 3LL cells selected for its high lung colonization potential was grown continuously in vitro for 24 weeks. The present study was undertaken to characterize the expression of both urokinase-type plasminogen activator (uPA) and urinary trypsin inhibitor (UTI) in the non-metastatic (3LL(-)) and the metastatic (3LL(+)) cells. Both cells were tested on the Matrigel for invasive ability using a modified Boyden chamber and assayed for expression of uPA and UTI. The 3LL(+) cells secreted 5 times more uPA (6.25 mu g per 10(6) cells per 24 h) than the 3LL(-) cells (1.25 mu g per 10(6) cells per 24 h). The 3LL(+) cells, which expressed 2 times more cell-surface receptor-bound enzymatically active uPA (0.32 +/- 0.06 OD405) than the 3LL(-) cells (0.15 +/- 0.03 OD405), had larger amounts of cell-surface receptor-bound uPA. On the other hands, UTI levels in the conditioned media was decreased 25-fold in the 3LL(+) cells (0.05 mu g/10(6) cells/24 h) compared to the 3LL(-) cells (1.25 mu g/10(6) cells/24 h). The 3LL(-) cells expressed significantly higher levels of cell-associated UTI as indicated by a cell ELISA (3LL(+), 0.30 +/- 0.04 OD450; 3LL(-), 1.30 +/- 0.21 OD450) and by Western blot analysis. Metastatic competence in the 3LL(+) tumor model is associated with increased expression and release of uPA, as well as decreased UTI production, consistent with a more invasive phenotype. These data support our hypothesis that UTI may contribute to the inhibition of uPA expression in tumor cells.

No effect of high-protein food on the stereoselective bioavailability and pharmacokinetics of verapamil.

The effects of high-protein food on the bioavailability of both the racemate and individual enantiomers of verapamil were investigated in 12 healthy volunteers using a randomized crossover design. Food had no effect on any parameter of bioavailability for both the racemate and the individual enantiomers of verapamil except time to maximum concentration (tmax), which was significantly prolonged after food intake. The pharmacokinetics of the enantiomers of norverapamil were not significantly changed by food intake. These results suggest that high-protein food does not alter the pharmacokinetics and bioavailability of either the racemate or the individual enantiomers of verapamil. Therefore, the clinical efficacy of verapamil is not related to food intake, except for a slight prolongation in the time to onset of the pharmacologic effects. The present data can be applied to the high-protein content meal intake.

A walking stick for a pure akinesia patient.

We describe a 63-year-old female patient with pure akinesia whose gait was facilitated by a handmade converted walking stick. A posterior ventral pallidotomy had been performed, but it did not alleviate symptoms. Her husband made a walking stick with a wire loop at the bottom, perpendicular to the walking direction. When the patient stepped over the loop, the frozen gait was improved. This converted walking stick is easily made and inexpensive. Although the walking stick did not improve the patient's gait radically, use of the converted walking stick effectively improved the patient's daily life because successful treatment of pure akinesia cannot be established.

Involvement of platelet-activating factor (PAF) in glutamate neurotoxicity in rat neuronal cultures.

To clarify the role of platelet-activating factor (PAF) in glutamate neurotoxicity, in vitro experiments using primary neuronal cultures were performed. The anti-PAF immunoglobulin-G (aPAF-IgG) and the three PAF receptor antagonists (BN52021, CV6209, and E5880) were tested for their neuroprotective activity in primary neuronal cultures isolated from embryonic rat cerebral cortex. The cultured neurons were exposed to glutamate (1 mM) for 60 min. Twenty-four hours after this exposure, aPAF-IgG demonstrated evidence of protective effects against neuronal damage in a dose-dependent manner. Protective effects also were observed in cultures treated with the three PAF antagonists (P < 0.05 at 1 microg/ml aPAF-IgG, P < 0.01 at 100 microM BN52021, P < 0.05 at 10 nM CV6209 and P < 0.01 at 10 nM E5880). The Fura-2 assay was used to estimate whether low dosages of exogenous PAF affect cultured neurons. The cultured neurons were loaded with Fura-2/AM. After preincubation for 120 min, the Fura-2-loaded neurons were exposed to various concentrations of PAF for 60 min. By measuring the fluorescent intensity of the medium as representing the amount of Fura-2 released from damaged neurons, we detected an increased release of Fura-2, even at low doses of PAF (P < 0.01 at 10 nM PAF). We further studied PAF production by neurons in response to glutamate. The level of PAF measured in the medium exposed to glutamate was significantly higher than the level in the medium unexposed to glutamate (P < 0.05). Our results suggest an important role of PAF in glutamate neurotoxicity.

In vitro hypoxia of cortical and hippocampal CA1 neurons: glutamate, nitric oxide, and platelet activating factor participate in the mechanism of selective neural death in CA1 neurons.

We prepared neuron-rich cultures from cortical and hippocampal CA1 regions of postnatal day 1 (P1) rats. Using these cultures, we investigated the sensitivity of neurons to hypoxic insults. The effects of MK-801, cycloheximide, NG-nitro-L-arginine (L-NNA), and anti-platelet-activating factor (anti-PAF) IgG on neuronal injury under hypoxic conditions also were examined. The percentage of astroglial cells was higher in CA1 than cortical cultures despite use of the same culture procedure. Despite this finding, the percentage of lactate dehydrogenase (LDH) released into the medium was greater in CA1 than cortical cultures under the conditions of 24-h hypoxia and 24-h incubation (P < 0.05). We then added MK-801 (500 nM), cycloheximide (3 microM), L-NNA (100 microM) and anti-PAF IgG (50 micrograms/ml) prior to inducing the hypoxia and measured LDH in the medium after 24-h hypoxia and 48-h incubation. Under the hypoxic condition, MK-801, L-NNA, and anti-PAF IgG significantly protected the CA1 neurons from hypoxic injury compared with cortical neurons, while cycloheximide protected both cultures equally. These results suggest that CA1 neurons are more sensitive to hypoxia than cerebral cortical neurons, and glutamate, nitric oxide, and PAF may participate in the mechanism of selective neural death in neurons of the CA1 region due to hypoxia.