Loss of Autophagy Disrupts Stemness of Ameloblast-Lineage Cells in Aging.

Autophagy is one of the intracellular degradation pathways and maintains cellular homeostasis, regulating the stress response, cell proliferation, and signal transduction. To elucidate the role of autophagy in the maintenance of dental epithelial stem cells and the subsequent enamel formation, we analyzed autophagy-deficient mice in epithelial cells (Atg7f/f;KRT14-Cre mice), focusing on the influence of aging and stress environments. We also performed in vitro cell and organ culture experiments with an autophagy inhibitor. In young Atg7f/f;KRT14-Cre mice, morphological change was not obvious in maxillary incisors, except for the remarkable cell death in the stratum intermedium of the transitional stage. However, under stress conditions of hyperglycemia, the incisor color changed to white in diabetes Atg7f/f;KRT14-Cre mice. Regarding dental epithelial stem cells, the shape of the apical bud region of the incisor became irregular with age, and odontoma was formed in aged Atg7f/f;KRT14-Cre mice. In addition, the shape of apical bud culture cells of Atg7f/f;KRT14-Cre mice became irregular and enlarged atypically, with epigenetic changes during culture, suggesting that autophagy deficiency may induce tumorigenesis in dental epithelial cells. The epigenetic change and upregulation of p21 expression were induced by autophagy inhibition in vivo and in vitro. These findings suggest that autophagy is important for the regulation of stem cell maintenance, proliferation, and differentiation of ameloblast-lineage cells, and an autophagy disorder may induce tumorigenesis in odontogenic epithelial cells.

Simple modification to improve reliability of copro-DNA examinations for diagnosing Echinococcus multilocularis infections in red foxes.

Epidemiological studies of Echinococcus multilocularis infections in definitive hosts require a reliable and economic diagnostic method. In this study, the current copro-DNA examination technique was modified by increasing the faecal amounts tested and adding a step to neutralize the faeces before DNA extraction. Reliability of the modified method was evaluated using rectal faecal samples from red foxes and comparing them with intestinal worms detected using the sedimentation and counting technique (SCT) following necropsy. The modified copro-DNA examination method demonstrated 93.9% sensitivity (138/147) on the SCT. Its detectability increased depending on the worm burden, and the sensitivity was 100% in cases harbouring over 1000 worms. From 111 SCT-negative cases, six (5.4%) were copro-DNA-positive, and all were confirmed as E. multilocularis via sequencing analysis. Five of the remaining 105 SCT-negative cases (4.8%) retained polymerase chain reaction (PCR) inhibitors in the extracted solution, suggesting that approximately 5% of the red fox faeces retained these inhibitors after treatment with the present copro-DNA extraction method. Although further evaluation is needed for faeces deposited in the wild, the present copro-DNA examination technique will help monitor the E. multilocularis prevalence in definitive hosts. When used for detailed evaluations of endemicity (e.g. changes in infection pressure or spread in non-endemic areas), the absence of PCR inhibitors should be confirmed, and multiple trials on faecal subsamples are recommended.

Regression of gastric de novo diffuse large B-cell lymphoma following Helicobacter pylori eradication: a case report.

We report a case of primary gastric diffuse large B-cell lymphoma (DLBCL), de novo DLBCL without the features of mucosa-associated lymphoid tissue (MALT) lymphoma, which regressed after Helicobacter pylori (HP) eradication. A 27-year-old Japanese female with epigastralgia was revealed to have ulcerated lesions in the angle and antral regions on gastroscopy. Biopsy specimen was consistent with a diagnosis of DLBCL without MALT lymphoma component, indicating de novo development. Her clinical staging on the Lugano system was Stage I. HP was positive on a rapid urease test, and she received HP eradication therapy twice, because the first therapy was not successful. On gastroscopy performed 1 month after the second HP eradication therapy, no ulcerated lesion was noted, and the lymphoma cells had regressed histopathologically. (Acta gastro-enterol. belg., 2016, 79, 367-369A).

The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis.

Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n= 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.

Differences in the developmental origins of the periosteum may influence bone healing.

BACKGROUND AND OBJECTIVE: The jaw bone, unlike most other bones, is derived from neural crest stem cells, so we hypothesized that it may have different characteristics to bones from other parts of the body, especially in the nature of its periosteum. The periosteum exhibits osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. MATERIAL AND METHODS: Gene expression profiles of jaw bone and periosteum were evaluated by DNA microarray and real-time polymerase chain reaction. Furthermore, we perforated an area 2 mm in diameter on mouse frontal and parietal bones. Bone regeneration of these calvarial defects was evaluated using microcomputed tomography and histological analysis. RESULTS: The DNA microarray data revealed close homology between the gene expression profiles within the ilium and femur. The gene expression of Wnt-1, SOX10, nestin, and musashi-1 were significantly higher in the jaw bone than in other locations. Microcomputed tomography and histological analysis revealed that the jaw bone had superior bone regenerative abilities than other bones. CONCLUSION: Jaw bone periosteum exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material to repair bone defects. A full investigation of the biological and mechanical properties of jaw bone as an alternative graft material for jaw reconstructive surgery is recommended.

Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma.

Cancer stem cells (CSCs) are among the target cells of cancer therapy because they are uniquely involved in both cancer progression and sensitivity to chemotherapeutic agents. We identified side population (SP) cells, which are known to be an enriched population of CSC, in five oral squamous cell carcinoma (OSCC) cells (SCC9, SCC25, TOSCC7, TOSCC17, and TOSCC23). The percentages of SP cells ranged from 0% to 3.3%, with TOSCC23 cells showing the highest percentages of SP cells (3.3% of the total cell population). The SP cells isolated from TOSCC23 cells also showed greater cell proliferation and invasion compared to non-SP (MP) cells. Therefore, our initial findings suggested that SP cells were enriched for CSC-like cells. Furthermore, DNA microarray analysis revealed that the expression of cell proliferation-related and anti-apoptotic genes was greater in SP cells compared to MP cells. We focused on Lin28a, which showed the highest expression (approximately 22-fold) among the upregulated genes. The overexpression of Lin28a in TOSCC23 cells increased their proliferation, colony formation, and invasion. These findings suggest that Lin28a is an appropriate CSC target molecule for OSCC treatment.

Surgical strategies for hepatocellular carcinoma with special reference to anatomical hepatic resection and intraoperative contrast-enhanced ultrasonography.

Here we described our strategies to attain a better prognosis for hepatocellular carcinoma (HCC) patients by surgery. Among a variety of attempts conducted to date, we focused on anatomical resection and intraoperative contrast-enhanced ultrasonography. There are still controversies with respect to the significance of anatomical resection. We analyzed the significance of this surgical procedure in 207 patients without macrovascular invasion. These patients underwent either anatomical resection or non-anatomical resection. We found that the patients with anatomical resection had higher recurrence-free survival rate than those with non-anatomical resection. Univariable analysis showed that liver damage, the serum level of alpha-fetoprotein, tumor number, surgical margin, and type of surgery (anatomical or non-anatomical resection) were significant predictive factors for intrahepatic recurrence. Multivariable analysis revealed that multiple tumors, alpha-fetoprotein, and non-anatomical resection were independent risk factors for recurrence. We conclude that anatomical resection is a recommendable surgical procedure in patients without macrovascular invasion. A recent innovation is the development of contrast-enhanced ultrasonography. Then we have applied this to liver surgery intraoperatively. We confirm that vascular images contribute to a precise diagnosis and the detection of small portal tumor thrombi, and that Kupffer images are useful to discover the minute tumors. In addition, by clarifying the relationship between tumors and the vascular architecture, real-time 3-dimensional images using Kupffer imaging are a promising guide during the surgical procedures, although further development is needed.

Cost benefit and clinical efficacy of low-dose granulocyte colony-stimulating factor after standard chemotherapy in patients with non-Hodgkin's lymphoma.

High costs of molecule-targeted drugs, such as rituximab, ibritumomab, and tositumomab have given rise to an economical issue for treating patients with non-Hodgkin's lymphoma (NHL). Granulocyte colony-stimulating factors (G-CSFs), which are also expensive, are widely used for treating neutropenia after chemotherapy. In Japan, lenograstim at 2 microg/kg (about 100 microg/body) or filgrastim at 50 microg/m(2) (about 75 microg/body) is commonly administered for patients with NHL after chemotherapy. Therefore, cost-effectiveness is an important issue in treatment for NHL. Patients with advanced-stage NHL who needed chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or a CHOP-like regimen with or without rituximab were enrolled in this randomized cross-over trial to investigate the efficacy and safety of low-dose G-CSF. Half of the patients were administered 75 microg filgrastim in the first course after neutropenia and 50 microg lenograstim in the second course, and the other half were crossed over. Forty-seven patients were enrolled in this cross-over trial, and 24 patients completed the trial. Frequencies and durations of grade 4 leukocytopenia and neutropenia were similar in the two groups. Severe infection was rare and was observed at similar frequency. Frequencies of antibiotics use were also similar. The total cost of G-CSF (cost/drug x duration of administration) was significantly lower in patients who received 50 microg lenograstim. Hence, a low dose of lenograstim might be safe, effective and pharmaco-economically beneficial in patients with advanced-stage NHL.

[Lung abscess which ruptured during the medical treatment of lung abscess; report of a case].

63-year-old man was admitted to our hospital with fever and cough for about 2 months. Laboratory data showed marked inflammatory changes, and chest computed tomography (CT) scans revealed right-sided hydrothorax, atelectasis of the right middle lobe, and a cystic mass in the right middle lobe. We diagnosed the patients as having lung abscess and empyema. Following the intravenous antibiotic chemotherapy, symptoms and laboratory data showed the improvement, however, on the 11th hospital day, he developed high fever again. A chest CT showed pneumopyothorax suggesting the rupture of lung abscess. Since the chest tube drainage was ineffective, open chest surgery was performed. Curettage of both thoracic and abscess cavity with closure of air leakage successfully cured the pyothorax.

The role of sex hormones in the kinetics of chondrocytes in the growth plate. A study in the rabbit.

Sex hormones play important roles in the regulation of the proliferation, maturation and death of chondrocytes in the epiphyseal growth plate. We have investigated the effects of male castration on the cell kinetics of chondrocytes as defined by the numbers of proliferating and dying cells. The growth plates of normal rabbits and animals castrated at eight weeks of age were obtained at 10, 15, 20 and 25 weeks of age. Our study suggested that castration led to an increase in apoptosis and a decrease in the proliferation of chondrocytes in the growth plate. In addition, the number of chondrocytes in the castrated rabbits was less than that of normal animals of the same age.

Expression of protein kinases C betaI, betaII, and VEGF during the differentiation of enamel epithelium in tooth development.

Protein kinase C (PKC) is an important molecule involved in various cell function, and mediates induced secretion of vascular endothelial growth factor (VEGF). It is hypothesized that PKC and VEGF may be associated with tooth development. Using the laser microdissection method and real-time reverse-transcription-polymerase chain-reaction (RT-PCR), we investigated the expression of PKC betaI and betaII, VEGF, and amelogenin (used as a marker of differentiation to ameloblasts) in the inner and outer enamel epithelia, stellate reticulum, and dental papilla in each stage of the dental germ. We found that the expression levels of PKC betaI and betaII were increased in the inner enamel epithelium during the early bell stage. In addition, the increased expression levels of PKC betaI and betaII were accompanied by increased VEGF expression. These results indicate that PKC betaI, betaII, and VEGF are closely associated with the differentiation of the inner enamel epithelium to ameloblasts.

Gene expression profiling of mouse condylar cartilage during mastication by means of laser microdissection and cDNA array.

Little is known about the mechanisms of mandibular condylar growth. In this study, gene expression in the mandibular condylar cartilage of young post-natal mice was monitored by means of a cDNA microarray, real-time PCR, and laser microdissection before and after the initiation of mastication (newborn, 7 days, 21 days, initiation of mastication, and 35 days). Insulin-like growth factor-1 (IGF-I), transforming-growth-factor-beta-2 (TGFbeta2), and aggrecan mRNAs were clearly expressed at 21 days, while the expression of osteopontin mRNAs was most clear at 35 days. Parathyroid-hormone-related protein (PTHrP), Indian-hedgehog (Ihh), and insulin-like growth factor-2 (IGF-2) mRNAs were clearly expressed during lactation (newborn and 7 days). Heat-shock-protein 84 (HSP-84) and heat-shock-protein 86 (HSP-86) were clearly expressed at 35 days. These results revealed that gene expression changed during mandibular condylar cartilage growth, and that, interestingly, these changes coincided with the initiation of mastication.

Serial Analysis of Gene Expression (SAGE) of Magnaporthe grisea: genes involved in appressorium formation.

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.

Protective effect of sulfobutyl ether beta-cyclodextrin on DY-9760e-induced hemolysis in vitro.

The hemolytic behavior of a novel cytoprotective agent, DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) was investigated using rabbit erythrocytes. Further, the effects of water-soluble cyclodextrin derivatives, such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether of beta-cyclodextrin (SBE-beta-CyD), on the hemolytic activity of DY-9760e were studied. DY-9760e induced hemolysis at concentrations >0.2-0.3 mM in phosphate buffered saline (PBS) of pH 4.0 and 6.0, where DY-9760e is predominantly in dicationic and monocationic forms, respectively. The hemolytic activity of the monocationic DY-9760e was higher than that of the dicationic species, and the hemolysis at pH 4.0 involved the formation of methemoglobin. DY9760e induced the morphological change of erythrocytes towards membrane invagination at both pH 4.0 and 6.0. SBE7-beta-CyD significantly suppressed the DY-9760e-induced hemolysis and morphological change at both pH 4.0 and 6.0, as well as the formation of methemoglobin at pH 4.0. On the other hand, HP-beta-CyD suppressed only the hemolysis, but neither the morphological change nor the formation of methemoglobin. In addition, the inhibitory effect of SBE7-beta-CyD on the hemolysis was greater than that of HP-beta-CyD. The superior inhibitory effect of SBE7-beta-CyD on the DY-9760-induced hemolysis, the morphological change, and the formation of methemoglobin may be attributable to the formation of a stable inclusion complex with DY-9760e and to the weaker hemolytic activity of SBE7beta-CyD than HP-beta-CyD. These results suggest potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.

Protective effects of estradiol against amyloid beta protein-induced inhibition of neuronal Cl(-)-ATPase activity.

Low concentrations of amyloid beta proteins (Abetas, 1-10 nM) were recently demonstrated to reduce Cl(-)-ATPase activity in parallel with an increase in the intracellular Cl(-) concentration ([Cl(-)]i) and decreases in plasma membrane phosphorylated phosphatidylinositol (PIP and PIP2) levels in cultured rat hippocampal neurons. In this study, 17 beta-estradiol (estradiol) at a therapeutic concentration (1.8 nM) for Alzheimer's disease was found to block these Abeta (Abeta25-35)-induced changes. This protective effect of estradiol on Cl(-)-ATPase activity was antagonized by a pure estrogen receptor antagonist, ICI182780 and inhibitors for cyclic GMP-dependent protein kinase (PKG) (KT5823), Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) (KN62) and phosphatidylinositol (PI) 4-kinase (wortmannin and quercetin). Estradiol recovered Abeta-induced decreases in plasma membrane phosphoinositide (PIP and PIP2) levels, this effect being inhibited by KT5823 and KN62. Glutamate toxicity was augmented in neurons with elevated [Cl(-)]i either by Abeta-treatment or carbachol+KCl+LiCl-treatment. The increased glutamate toxicity in the Abeta-treated neurons was attenuated by estradiol. Thus, a therapeutic concentration of estradiol protected Abeta-treated neurons against inhibition of Cl(-)-ATPase activity and an increase in [Cl(-)]i through its receptor, probably via PKG- and CaMKII(-)mediated recovery of PI4P formation. Elevated [Cl(-)]i may be related to enhancement of glutamate toxicity.

CT fluoroscopy-guided intervention: marked reduction of scattered radiation dose to the physician's hand by use of a lead plate and an improved I-I device.

PURPOSE: To estimate the effects of a lead plate, three types of needle holders, tube current, and slice thickness on decreasing the radiation dose to the physician's hand during interventional procedures with computed tomographic (CT) fluoroscopic guidance. The needle holders (I-I devices), which were developed by the authors, maintained the distance between the physician's hand and the CT plane at 7 cm, 10 cm, and 15 cm, respectively. MATERIALS AND METHODS: The dose rate (mSv/tube current/CT fluoroscopy time) was measured in 55 cases, which were divided into six groups. In group A (n = 14), the current was 135 kV, there was a 5-mm slice thickness, and a 7-cm I-I device was used without the lead plate. Group B (n = 11) entailed a 120-kV current, a 5-mm slice thickness, and a 7-cm I-I device without the lead plate. Group C (n = 8) entailed a 120-kV current, 5-mm slice thickness, and 7-cm I-I device with the lead plate. Group D (n = 9) entailed a 120-kV current, 5-mm slice thickness, and 10-cm I-I device with the lead plate. Group E (n = 7) entailed a 120-kV current, 5-mm slice thickness, and 15-cm I-I device with the lead plate. Group F (n = 6) entailed a 120-kV current, 1-mm slice thickness and 10-cm I-I device with the lead plate. To compare the effects of tube voltage, lead plate use, slice collimation, and I-I devices, differences were compared between groups A and B, B and C, D and F, and among groups C, D, and E. RESULTS: The dose rates of groups A, B, C, D, E, and F were 126.3, 75.2, 17.8, 13.9, 2.8, and 4.1 mSv/mA/sec x 100,000, respectively. There were significant differences in dose rates between groups A and B (t-test, P =.037), B and C (Student t-test, P =.002), D and F (Mann-Whitney test, P =.011), and among groups C, D, and E (Kruskal-Wallis test, P =.016). CONCLUSION: The lead plate, the improved I-I devices, use of a 120 kV (vs 135 kV) current, and 1-mm (vs 5 mm) collimation were all useful in decreasing the dose rate.

Dose-finding phase I study of simultaneous weekly infusion with doxorubicin and docetaxel in patients with advanced breast cancer.

BACKGROUND: Combination therapy with doxorubicin (DOX) and docetaxel (DOC), given 3 weeks apart, is one of the standard regimens used for treating metastatic breast cancer, but it frequently generates febrile neutropenia. To find a safer regimen with less myelotoxicity and the appropriate dose intensity, we conducted a phase I study of simultaneous weekly infusion with DOX and DOC. METHODS: Twenty-five patients with advanced breast cancer were treated with an intravenous push-injection of DOX that was immediately followed by a 1-h infusion of DOC. This was repeated every week for at least 6 weeks. The premedication employed was three 4-mg doses of dexamethasone every week. Patients were divided into four groups for which the doses of DOX and DOC were escalated in 5-mg/m2 increments. RESULTS: In the 18 patients who were treated with DOX 15 or 20 mg/m2 and DOC 25 mg/m2, or lower, the regimen was found to be tolerable, without febrile episodes. The regimen with 20 mg/m2 of DOX and 30 mg/m2 of DOC was the maximum tolerated dose. Other indications of grade 3 toxicity included asthenia in 4% of patients, anorexia in 8%, and vomiting in 8%. Of the 25 patients, 14 had a partial response. The overall response rate was 56% (95% confidence interval [CI], 35% to 77%). The recommended dose for further trial was 20 mg/m2 of DOX and 25 mg/m2 of DOC. CONCLUSION: Simultaneous weekly infusion with DOX and DOC was feasible, with modest neutropenia and preserved dose intensity. This regimen may be helpful in the management of patients with advanced breast cancer.

Improvement of some pharmaceutical properties of DY-9760e by sulfobutyl ether beta-cyclodextrin.

The interaction of DY-9760e, a novel cytoprotective agent, with sulfobutyl ether beta-cyclodextrin (SBE-beta-CyD) in phosphate buffered saline (PBS) at various pH and ionic-strengths was studied by spectroscopic methods and the solubility method, and the results were compared with that of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD). The circular dichroism (CD) spectroscopic studies suggested that both beta-CyDs form the inclusion complexes with DY-9760e in a molar ratio of 1:1, and the interaction of DY-9760e with SBE-beta-CyD is much stronger than that with HP-beta-CyD at any pH studied, in terms of a synergetic effect of hydrophobic and electrostatic interactions. The different intermolecular interaction between the SBE-and HP-beta-CyD complexes was clearly reflected in the stability constant (K'), e.g. the different dependence of K' value on pH and ionic strength of solutions. 1H- and 13C-NMR studies suggested that HP-beta-CyD interacts preferably with the benzene ring of DY-9760e, whereas SBE-beta-CyD interacts not only with the benzene ring via hydrophobic interaction but also with the piperazine ring of the drug via electrostatic interaction. The solubilizing ability of SBE-beta-CyD against DY-9760e was much greater than that of HP-beta-CyD at any pH studied. Furthermore, SBE-beta-CyD markedly suppressed the photo-degradation of DY-9760e in aqueous solution and reduced the adsorption of DY-9760e from PBS to polyvinyl chloride (PVC) tubes after incubation. The results suggest that SBE-beta-CyD is useful in preparing parenteral solutions of poorly water-soluble drugs with positive charge such as DY-9760e.

Intraarterial chemotherapy of liver metastases: implantation of a microcatheter-port system with use of modified fixed catheter tip technique.

A new method was developed to implant a microcatheter-port system for repeat intraarterial chemotherapy of liver metastases. The microcatheter-port system was successfully implanted in all 20 patients reported in this study, and the only complications were one early occlusion of the hepatic artery and one dislocation of the implanted catheter.

Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons.

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.