An atomically controlled Si film formation process at low temperatures using atmospheric-pressure VHF plasma.

To grow epitaxial Si films with atomic- and electronic-level perfection, a high-temperature chemical vapor deposition (CVD) process (>1000 °C) has been generally employed. To reduce the growth temperature below 600 °C but keeping a high deposition rate, other energy sources than thermal heating are required. Atmospheric pressure plasma CVD (AP-PCVD) is considered to be suitable for fabricating high-quality films at high deposition rates due both to the high radical density and to the low ion bombardment against the film surface, because the collision frequency among ions and neutral atoms is high. The present study focuses on the low-temperature growth of epitaxial Si, and experimentally demonstrates that AP-PCVD is capable of growing epitaxial Si films with high perfection applicable for semiconductor devices. It is found that the pre-growth cleaning of the Si surface by H(2) AP plasma is effective to grow high-purity Si films, and that the exposure of a film-growing surface to AP plasma during growth is important to form particle-free and defect-free Si films. From the experimental results and the first-principles molecular dynamics simulations of surface atomic reactions, it can be mentioned that both H atoms in the AP plasma and high-density He atoms having thermal kinetic energy contribute to the reduction of growth temperature by supplying considerable energy to the surface.

Expression profiles of genes associated with viral entry in HCV-infected human liver.

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV-infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL-cholesterol (LDL-C) and HCV core antigen were also evaluated, and expression of claudin-1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin-1 genes was significantly suppressed (P < 0.0001) and occludin transcription was significantly up-regulated in HCV-infected livers (P < 0.0001). Significant positive correlations were found for LDLR versus occludin, LDLR versus claudin-1, occludin versus claudin-1, and CD81 versus SR-BI in HCV-infected (P = 0.0012, P < 0.0001, P = 0.0004, and P < 0.0001, respectively) and normal livers (P < 0.0001, P = 0.0051, P < 0.0001, and P < 0.0001, respectively). Positive correlation was observed between serum levels of HCV core antigen and LDL-C (P = 0.0147), with their levels negatively correlated to LDLR (P = 0.0270 and P = 0.0021, respectively). Immunohistochemically, hepatocellular expression of claudin-1 and occludin was increased in HCV-infected livers. Different levels of expression were demonstrated at the mRNA and protein levels for occludin and claudin-1 in HCV-infected and normal livers. Correlation of elements associated with viral entry was comparable in HCV-infected and normal livers.

Therapeutic effect of bezafibrate against biliary damage: a study of phospholipid secretion via the PPARalpha-MDR3 pathway.

OBJECTIVE: Bezafibrate (BF) has been used to treat biliary damage, particularly in patients with primary biliary cirrhosis (PBC), and its clinical efficacy has been demonstrated. The mechanism of action is thought to involve activation of the PPARalpha-MDR3-phospholipid (PL) secretion pathway. We tried to confirm this hypothesis in patients with hepatobiliary disease. METHODS: The levels of serum gamma-glutamyl transpeptidase and alkaline phosphatase, and those of bile components were examined before and after BF administration in patients with obstructive jaundice undergoing percutaneous transhepatic biliary drainage (PTBD). Hepatic expression of PPARalpha and MDR3 was quantified by real-time PCR in patients with PBC or non-alcoholic fatty liver disease (NAFLD). RESULTS: In patients with obstructive jaundice, BF decreased the serum levels of biliary enzymes and increased the bile concentration of PL. In patients with PBC or NAFLD, the expression levels of MDR3 were already up-regulated before starting the BF treatment. Although BF treatment did not further up-regulate MDR3 expression in NAFLD patients, PPARalpha expression was significantly increased. CONCLUSIONS: BF enhanced the secretion of PL into bile in cholestatic patients undergoing PTBD. However, in patients with PBC or NAFLD, diseases that represent cholesterol overload, MDR3 was already expressed at a high level to compensate for bile acids overproduction, and its expression was hardly affected by BF. In patients with chronic liver diseases such as PBC, BF may induce clinical effects via mechanisms independent of PL secretion.

Inhibition of protein phosphatase 2A overrides tau protein kinase I/glycogen synthase kinase 3 beta and cyclin-dependent kinase 5 inhibition and results in tau hyperphosphorylation in the hippocampus of starved mouse.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease (AD) brain. Starvation of adult mice induces tau hyperphosphorylation at many paired helical filaments sites and with a similar regional selectivity as those in AD, suggesting that a common mechanism may be mobilized. Here we investigated the mechanism of starvation-induced tau hyperphosphorylation in terms of tau kinases and Ser/Thr protein phosphatases (PP), and the results were compared with those reported in AD brain. During starvation, tau hyperphosphorylation at specific epitopes was accompanied by decreases in tau protein kinase I/glycogen synthase kinase 3 beta (TPKI/GSK3 beta), cyclin-dependent kinase 5 (cdk5), and PP2A activities toward tau. These results demonstrate that the activation of TPKI/GSK3 beta and cdk5 is not necessary to obtain hyperphosphorylated tau in vivo, and indicate that inhibition of PP2A is likely the dominant factor in inducing tau hyperphosphorylation in the starved mouse, overriding the inhibition of key tau kinases such as TPKI/GSK3 beta and cdk5. Furthermore, these data give strong support to the hypothesis that PP2A is important for the regulation of tau phosphorylation in the adult brain, and provide in vivo evidence in support of a central role of PP2A in tau hyperphosphorylation in AD.

Involvement of cyclin dependent kinase5 activator p25 on tau phosphorylation in mouse brain.

P35 or its truncated fragment p25 is required for cyclin dependent kinase (Cdk)5 activation. It has been reported that p25 is accumulated in the brain of Alzheimer's disease (AD) patients and that p25/Cdk5 induces high phosphorylation of tau and apoptosis in cultured neurons (Nature 402 (1999) 615). Our investigation of AD brain did not show specific accumulation of p25. Exposure to Ca ionophore (A23187) at 10(-6) M induced p25 accumulation in rat primary hippocampal neurons, causing neuronal death without showing hyperphosphorylation of tau. Transgenic mice expressing p25 showed the accumulation of p25 but neither hyperphosphorylation of tau nor neuronal death was shown in these mice. The feature of these mice was the progression of cell growth in pituitary gland. These results suggest that overexpression of p25 lead to the activation of cell cycle but not to the direct phosphorylation of tau.

Abeta amyloidosis induces the initial stage of tau accumulation in APP(Sw) mice.

To clarify how Abeta deposits induce secondary tauopathy, the presence of phosphorylated tau, glycogen synthase kinase 3alpha (GSK3alpha), GSK3beta, cyclin-dependent kinase 5 (CDK5), mitogen-activated protein kinase (MAPK) and fyn were examined in the Tg2576 brain showing substantial brain Abeta amyloidosis and behavioral abnormalities. Phosphorylated tau at Ser199, Thr231/Ser235, Ser396 and Ser413 accumulated in the dystrophic neurites of senile plaques. The major kinase for tau phosphorylation was GSK3beta. Smaller contributions of GSK3alpha, CDK5 and MAPK were suggested. Thus, brain Abeta amyloidosis has a potential role in the induction of tauopathy leading to the mental disturbances of Alzheimer's disease.

Familial Alzheimer's disease-associated mutations block translocation of full-length presenilin 1 to the nuclear envelope.

A polyclonal antibody, M5, to the hydrophilic loop domain of human presenilin 1 (PS1) was prepared. Western blot and immunoprecipitation analyses showed that M5 specifically recognized the processed C-terminal fragment, but not the full-length PS1. Epitope mapping analysis revealed that the essential sequence for recognition of the C-terminal fragment by M5 is DPEAQRR (302-308). The recognition of the C-terminal fragment by M5 in a processing-dependent manner was further confirmed by competitive enzyme-linked immunosorbent assay using the synthetic peptide L281 (281-311), which contains the putative processing site and the preceding amino acids to the site. Although L281 contains the epitope sequence for M5, the maximum inhibition was only 14%. Immunocytochemistry using M5 combined with hL312, which recognizes both full-length PS1 and the C-terminal fragment, allowed us to distinguish the localization of the processed C-terminal fragment from that of full-length PS1. Confocal microscopy demonstrated that the full-length form of wild-type PS1 is preferentially located in the nuclear envelope, while the processed C-terminal fragment is mainly present in the endoplasmic reticulum (ER). However, PS1 with familial Alzheimer's disease-associated mutations could not translocate to the nuclear envelope, and both the full-length and processed mutants were co-localized in the ER.

[A late phase II study of raltitrexed (ZD 1694) in chemotherapy-naive patients with advanced colorectal cancer].

A multicenter co-operative late phase II study of raltitrexed (ZD1694), a specific thymidylate synthase (TS) inhibitor, was conducted in chemotherapy-naive patients with advanced colorectal cancer. Raltitrexed was infused intravenously over 15 minutes once every three weeks. Between April 1996 and September 1998, 61 patients were enrolled and 58 were eligible. Fourteen patients experienced a partial response (PR), 22 no change (NC), 20 progressive disease (PD) and 2 no evaluable (NE). The overall response rate was 24.1% (95% CI: 13.9-37.2%). Responses were seen in lung (22.7%), liver (22.9%) and deep lymph nodes (10.0%). Median survival was 11.6 months. Grade 3 or 4 toxicities were: leukopenia (13.8%), neutropenia (24.1%), hemoglobin decrease (15.5%), FBC decrease (6.9%), hematocrit decrease (6.9%), thrombocytopenia (6.9%), transient SGPT increase (6.9%), nausea/vomiting (20.7%), anorexia (15.5%), and asthenia (6.9%). These adverse reactions were considered to be manageable. Only one death was associated with drug treatment. These results suggest that raltitrexed provides an effective and convenient treatment for patients with advanced and previously untreated colorectal cancer.

Lithium inhibits neurite growth and tau protein kinase I/glycogen synthase kinase-3beta-dependent phosphorylation of juvenile tau in cultured hippocampal neurons.

Tau protein kinase I(TPKI)/glycogen synthase kinase (GSK)-3beta is abundant in the developing rat brain. The highly phosphorylated juvenile form of tau is present during the same developmental period. To study the role of TPKI/ GSK-3beta in neuronal growth, we examined the effects of lithium, a direct inhibitor of TPKI/GSK-3beta, using primary cultures of rat hippocampal neurons. Immunohistochemical staining of the neurons indicates that in the presence of lithium (2-10 mM), neurite growth becomes inhibited in a dose-dependent manner. Western blot analyses of the cell extracts revealed that the presence of lithium in the culture medium increased the amount of dephosphorylated tau while decreasing phosphorylation at Ser199 and Ser396, both of which are TPKI/GSK-3beta phosphorylation sites on tau. The inhibition by lithium is reversible. Although the amount of TPKI/GSK-3beta remained constant, the amount of tau decreased in a dose-dependent manner in the presence of lithium. TPKI/GSK-3beta was distributed in the somata and proximal neurites of the cultured hippocampal neurons. These results therefore suggest that TPKI/GSK-3beta plays an important role in the axonal growth of neurons during synapse formation in the developing brain.

Dual roles of proteasome in the metabolism of presenilin 1.

Presenilin 1 (PS1) has been identified as a causative gene for most early-onset familial Alzheimer's disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild-type and familial Alzheimer's disease-associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full-length PS1 was significantly accumulated. The levels of N- and C-terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full-length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met288 and Glu299. Metabolic labeling experiments showed that the appearance of the N-terminal fragment was attenuated by the inhibitor. Finally, 28-kDa N- and 20-kDa C-terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.

Activation of tau protein kinase I/glycogen synthase kinase-3beta by amyloid beta peptide (25-35) enhances phosphorylation of tau in hippocampal neurons.

According to the amyloid hypothesis for the pathogenesis of Alzheimer's disease (AD), amyloid beta peptide (Abeta) directly affects neurons, leading to neurodegeneration and tau phosphorylation, followed by the production of paired helical filaments (PHF) in neurofibrillary tangles (NFT). To analyze the relationship between the phosphorylation sites of tau and the activation of kinases in response to Abeta, we treated cultured rat hippocampal neurons with a peptide fragment of Abeta, Abeta(25-35). Abeta(25-35) treatment activated tau protein kinase I/glycogen synthase kinase-3beta (TPKI/GSK-3beta) but not glycogen synthase kinase-3alpha (GSK-3alpha) or mitogen activated protein kinase (MAP kinase) in primary culture of hippocampal neurons. Using antibodies that recognize phosphorylated sites of tau, we showed that tau phosphorylation was enhanced in at least five sites (Ser199, Ser202, Ser396, Ser404, and Ser413 numbered according to the human tau isoform containing 441 amino acid residues), to an extent that depended on the level of TPK I/GSK-3beta. Treatment with TPK I/GSK-3beta antisense oligonucleotide inhibited the enhancement of tau phosphorylation induced by Abeta(25-35) exposure. Thus, TPK I/GSK-3beta activation by Abeta(25-35) may lead to extensive tau phosphorylation.

Direct association of presenilin-1 with beta-catenin.

Families bearing mutations in the presenilin-1 (PSI) gene develop Alzheimer's disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co-immunoprecipitation with PS1 in transfected COS-7 cells indicates that PSI directly interacts with endogenous beta-catenin, and the interaction requires residues 322450 of PSI and 445-676 of beta-catenin. Both proteins are co-localized in the endoplasmic reticulum. Over-expression of PS1 reduces the level of cytoplasmic beta-catenin, and inhibits beta-catenin-T cell factor-regulated transcription. These results indicate that PSI plays a role as inhibitor of the beta-catenin signal, which may be connected with the AD dysfunction.

Presenilin 1 associates with glycogen synthase kinase-3beta and its substrate tau.

Families bearing mutations in the presenilin 1 (PS1) gene develop Alzheimer's disease. Previous studies have shown that the Alzheimer-associated mutations in PS1 increase production of amyloid beta protein (Abeta1-42). We now show that PS1 also regulates phosphorylation of the microtubule-associated protein tau. PS1 directly binds tau and a tau kinase, glycogen synthase kinase 3beta (GSK-3beta). Deletion studies show that both tau and GSK-3beta bind to the same region of PS1, residues 250-298, whereas the binding domain on tau is the microtubule-binding repeat region. The ability of PS1 to bring tau and GSK-3beta into close proximity suggests that PS1 may regulate the interaction of tau with GSK-3beta. Mutations in PS1 that cause Alzheimer's disease increase the ability of PS1 to bind GSK-3beta and, correspondingly, increase its tau-directed kinase activity. We propose that the increased association of GSK-3beta with mutant PS1 leads to increased phosphorylation of tau.

[A cooperative study on concomitant with low-dose divided administration of cisplatin (CDDP) and sustained drip infusion of 5-fluorouracil (5-FU) for unresectable advanced gastric cancer. Osaka Cisplatin Gastric Cancer Study Group].

For patients with unresectable gastric cancer registered between July 1994 and September 1995, the following dosage regimen was examined: a drip infusion of cisplatin (CDDP) at 7 mg/m2/day for 5 consecutive days and 2-day withdrawal a week for 3 weeks with concomitant sustained drip infusion of 5-FU at 300 mg/m2/day for 21 days. The whole cycle was repeated again after 2-week withdrawal. The antitumor effect was seen in 39.4% among 33 cases. With respect to histological classification, there was no difference in appearance of the effect between the moderately differentiated type at 62.5% and the low-differentiated type at 42.9%. The cases which became resectable after the administration included those in which the tumor disappeared completely in the resected specimens. As adverse reactions encountered in the cases above Grade 3, anorexia, nausea and vomiting and diarrhea were seen as clinical symptoms in 10, 5.3 and 11.8%, respectively. As laboratory test values, decrease in hemoglobin, leukocytopenia and thrombocytopenia were seen in 16.7, 15.8 and 5%, respectively, while renal or liver dysfunction did not occur. This dosage regimen was considered useful for unresectable advanced gastric cancer, although myelosuppression should be watched carefully.

Effects of changing the type of H2-blocker in the treatment of H2-blocker-resistant ulcers: comparison of roxatidine acetate hydrochloride and other H2-blockers.

The efficacy of switching from one type of H2-receptor antagonist (H2-blocker) to another, in the treatment of H2-blocker-resistant ulcers was investigated using H2-blockers with five-membered rings (five-membered-ring agents)--such as cimetidine, ranitidine and famotidine--and an H2-blocker with a six-membered ring, roxatidine. By switching from a five-membered-ring agent to roxatidine in the treatment of five-membered-ring resistant ulcers (study I), gastric ulcers were healed in nine of 19 patients (47%) and duodenal ulcers were healed in eight of nine patients (89%). By switching from roxatidine to one of the five-membered-ring agents in the treatment of roxatidine-resistant ulcers (study II), gastric ulcer was healed in six of 15 patients (40%), and duodenal ulcer was healed in 4 of 10 patients (40%). Particularly in the case of duodenal ulcers, the switch to treatment with roxatidine, which has a different chemical structure from the five-membered-ring agents, may be useful in the treatment of five-membered-ring-resistant ulcers.

Different effects of Alzheimer-associated mutations of presenilin 1 on its processing.

Presenilin 1 (PS 1) shows missense mutations in most early-onset familial Alzheimer's disease (FAD). Transfection of cDNA for wild type PS 1 into rat pheochromocytoma PC12 cells generated a 47 kDa full-size PS 1 protein, which was processed into a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment. We prepared selected Alzheimer-associated mutations (Gly384Ala, Leu392Val, and Cys410Tyr) of PS 1, which localized after a possible cleavage site. By transient expression in PC12 cells and rat glioma cell line, C6, we examined their influence on the processing of PS 1. Cys410Tyr inhibited proteolytic processing of PS 1, while Gly384Ala and Leu392Val did not. Thus, the Alzheimer related mutations can be divided into two groups in terms of their effect on the proteolytic cleavage of PS 1.

Nontoxic amyloid beta peptide 1-42 suppresses acetylcholine synthesis. Possible role in cholinergic dysfunction in Alzheimer's disease.

We show here that amyloid beta peptide1-42 (Abeta1-42) may play a key role in the pathogenesis of the cholinergic dysfunction seen in Alzheimer's disease (AD), in addition to its putative role in amyloid plaque formation. Abeta1-42 freshly solubilized in water (non-aged Abeta1-42), which was not neurotoxic without preaggregation, suppressed acetylcholine (ACh) synthesis in cholinergic neurons at very low concentrations (10-100 nM), although non-aged Abeta1-40 was ineffective. Non-aged Abeta1-42 impaired pyruvate dehydrogenase (PDH) activity by activating mitochondrial tau protein kinase I/glycogen synthase kinase-3beta, as we have already shown in hippocampal neurons (Hoshi, M., Takashima, A., Noguchi, K., Murayama, M., Sato, M., Kondo, S., Saitoh, Y., Ishiguro, K., Hoshino, T., and Imahori, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2719-2723). Neither choline acetyltransferase activity nor choline metabolism was affected. Therefore, the major cause of reduced ACh synthesis was considered to be an inadequate supply of acetyl-CoA owing to PDH impairment. Soluble Abeta1-42 increases specifically in AD brain (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem. 271, 4077-4081). This increase in soluble Abeta1-42 may disturb cholinergic function, leading to the deterioration of memory and cognitive function that is characteristic of AD.

Coexistence of choriocarcinoma and adenocarcinoma in the rectum: molecular aspects.

Choriocarcinoma, a malignant tumor of usually placental origin, in divided into two groups; the gestational and non-gestational types, the latter being rare. Non-gestational choriocarcinoma occurs in the lung, mediastinum, kidney, stomach, and small intestine, but rarely appears in the large intestine. We treated a 29-year-old woman with choriocarcinoma of the rectum with adenocarcinoma. Despite the rarity of the condition and the obscurity of the histogenesis, reports of similar cases and the occurrence of the tumors in the digestive tract suggest that the condition constitutes a clinical entity of a digestive tumor.

[Combined chemotherapy with MMC, ADM, CDDP, etoposide (VP-16) and 5'-DFUR, (MACVD therapy) as a second-line chemotherapy for metastatic gastric cancer: three cases].

Three patients with chemotherapeutically pretreated metastatic gastric cancer were given MAC-VD therapy combining MMC, ADM, CDDP, Etoposide (VP-16) and 5'-DFUR. All were evaluable for their responses. Patients ranged in age from 49 to 61 years. Performance status scale (P.S.) grade 0 was two cases; and P.S. grade 1 was one case. The overall response rate, CR+PR, was 0+2/3 (66.7%). The response rate in the primary lesions was 0%, against 66.7% (2/3) in the liver, 100% (1/1) in the spleen, 100% (1/1) in the lung, and 0% in the abdominal lymph node metastasis. The chief manifestations of toxicity were hematologic, such as leukocytopenia, anemia and thrombocytopenia in 100% of the cases. Non-hematologic toxicity was seen in alopecia in 66.7%, diarrhea in 33.3%, fever in 33.3%, and pigmentation in 33.3%. Severe toxicity was not observed. From these data, the administration of MAC-VD therapy was considered tolerable and these data suggested that this therapy could be given as a second-line chemotherapy when initial treatment failed to obtain a response after a partial response.

Combination therapy with cisplatin, 5'-deoxy-5-fluorouridine (5'-DFUR) and mitomycin (MMC) in patients with inoperable, advanced gastric cancer.

The optimal dose of cisplatin (CDDP) for combination chemotherapy for the treatment of inoperable, advanced gastric cancer has yet to be established. We therefore performed a randomized study to compare the therapeutic usefulness of two dose levels of cisplatin. 5'-deoxy-5-fluorouridine (5'-DFUR 1,400 mg/m(2)/d) was given orally on days 1 to 4 and 15 to 18. Mitomycin C (MMC, 5.75 mg/m(2)/d) was injected intravenously on day 5. In addition, 80 mg/m2/d of CDDP (regimen A) or 60 mg/m(2)/d of CDDP (regimen B) was given by 2-h intravenous drip infusion on day 5. This treatment cycle was repeated every four weeks. Fifty-six patients were enrolled. Clinical response was evaluated in 32 patients (regimen A, 16 patients; regimen B? 16 patients) with measurable lesions. The response rate was significantly higher with regimen A (9 PR/16, 56.3%) than with regimen B (3 PR/16, 18.9%) (p=0.028, chi(2) test). Median survival was slightly but not significantly longer with regimen A (7.4 months) than with regimen B (6.3 months). Drug toxicity included myelosuppression and gastrointestinal symptoms, but there were no serious adverse reactions or differences in safety between the treatment regimens. Regimen A was associated with a high response rate and low toxicity. The optimal dose of CDDP in combination with 5'-DFUR and MMC for the treatment of advanced gastric cancer is regarded to be 80 mg/m(2).