Gene therapy improves dental manifestations in hypophosphatasia model mice.

BACKGROUND AND OBJECTIVE: Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. MATERIAL AND METHODS: Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. RESULTS: Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. CONCLUSIONS: Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.

Thoracoscopic esophagectomy with extended lymph node dissection in the left lateral position: technical feasibility and oncologic outcomes.

The aim of this study was to estimate the technical and oncologic feasibility of video-assisted thoracoscopic radical esophagectomy (VATS) in the left lateral position. From January 2003 to December 2011, 132 patients with esophageal cancer underwent VATS. The mean duration of the thoracic procedure and the entire procedure was 294 ± 88 and 623 ± 123 minutes, respectively. Mean blood loss during the thoracic procedure and the entire procedure was 313 ± 577 and 657 ± 719 g, respectively. The mean number of dissected thoracic lymph nodes was 32.6 ± 12.9. There were four in-hospital deaths (3.0%); two patients (1.5%) died of acute respiratory distress syndrome and two patients (1.5%) died of tumor progression. Postoperative unilateral or bilateral recurrent laryngeal nerve (RLN) palsy, or pneumonia was found in 33 (25.0%), 21 (15.9%), and 27(20.5%) patients, respectively. The patients were divided into the first 66 patients who underwent VATS (Group 1) and the subsequent 66 patients (Group 2). The numbers of cases who underwent neoadjuvant or induction chemotherapy for T4 tumor and intrathoracic anastomosis were higher in Group 2 than in Group 1. The duration of the procedure, amount of blood loss, and the number of dissected thoracic lymph nodes were not different between the two groups. The total number of dissected lymph nodes was higher in Group 2 than in Group 1 (72.6 ± 27.8 vs. 62.6 ± 21.6, P = 0.023). The rate of bilateral RLN palsy was less in Group 2 than in Group 1 (7.6% vs. 24.2%, P = 0.042). The mean follow-up period was 38.7 months. Primary recurrence consisted of hematogenous, lymphatic, peritoneal dissemination, pleural dissemination, and locoregional in 15 (11.3%), 20 (15.1%), 3 (2.3%), 4 (3.0%), and 5 patients (3.8%), respectively. The rate of regional lymph node recurrence within the dissection field was only 4.5%. The prognosis of patients with lymph node metastasis was significantly poorer than that of patients without lymph node metastasis. However, the prognosis of the 11 cases that had metastasis only around RLNs was similar to that of node-negative cases. Thirteen patients with pathological remnant tumor (R1 or R2) did not survive longer than 5 years at present. The overall 5-year survival rate of stage I, II, and III disease after curative VATS was 82.2%, 77.0%, and 52.3%, respectively. Expansion of VATS criteria for patients after induction chemotherapy for T4 tumor or thoracoscopic anastomosis did not adversely affect the surgical results by experience. Although the VATS procedure is accompanied by a certain degree of morbidity including RLN palsy and pulmonary complications, VATS has an excellent locoregional control effect. In addition, the favorable survival after VATS shows that the procedure is oncologically feasible.

Changes in prostaglandin F2α receptor bindings in the hen oviduct uterus before and after oviposition.

The specific binding component for prostaglandin F(2α) (PGF(2α)) that exists in the plasma membrane fraction of the oviduct uterus myometrium of laying hens was shown to possess receptor properties for PGF(2α), such as binding specificity to PGF(2α), binding saturation, high affinity, and limited capacity. The value of the equilibrium dissociation constant (K(d)) for the receptor was not different between laying hens and nonlaying hens, but the value of the maximum binding capacity (B(max)) was smaller in laying hens than in nonlaying hens. During an oviposition cycle, the K(d) value did not show a significant change, but the B(max) value decreased at 3 and 0.5 h before oviposition and 2 h after oviposition. Neither the K(d) nor B(max) value changed in nonlaying hens during a 24-h period. An intravenous injection of PGF(2α) (5 µg/hen) decreased the B(max) value, but not the K(d) value, of the PGF(2α) receptor. It is thought from the results that PGF(2α) may act directly on the oviduct uterus myometrium at a fixed time before and after oviposition in laying hens.

Prospective randomized controlled study on the effects of perioperative administration of a neutrophil elastase inhibitor to patients undergoing video-assisted thoracoscopic surgery for thoracic esophageal cancer.

Sivelestat sodium hydrate (Ono Pharmaceutical Co., Osaka, Japan) is a selective inhibitor of neutrophil elastase (NE) and is effective in reducing acute lung injury associated with systemic inflammatory response syndrome (SIRS). We conducted a prospective randomized controlled study to investigate the efficacy of perioperative administration of sivelestat sodium hydrate to prevent postoperative acute lung injury in patients undergoing thoracoscopic esophagectomy and radical lymphadenectomy. Twenty-two patients with thoracic esophageal cancer underwent video-assisted thoracoscopic esophagectomy with extended lymph node dissection in our institution between April 2007 and November 2008. Using a double-blinded method, these patients were randomly assigned to one of two groups preoperatively. The active treatment group received sivelestat sodium hydrate intravenously for 72 hours starting at the beginning of surgery (sivelestat-treated group; n= 11), while the other group received saline (control group; n= 11). All patients were given methylprednisolone immediately before surgery. Postoperative clinical course was compared between the two groups. Two patients (one in each group) were discontinued from the study during the postoperative period because of surgery-related complications. Of the remaining 20 patients, 2 patients who developed pneumonia within a week after surgery were excluded from some laboratory analyses, so data from 18 patients (9 patients in each group) were analyzed based on the arterial oxygen pressure/fraction of inspired oxygen ratio, white blood cell count, serum C-reactive protein level, plasma cytokine levels, plasma NE level, and markers of alveolar type II epithelial cells. In the current study, the incidence of postoperative morbidity did not differ between the two groups. The median duration of SIRS in the sivelestat-treated group was significantly shorter than that in the control group: 17 (range 9-36) hours versus 49 (15-60) hours, respectively (P= 0.009). Concerning the parameters used for the diagnosis of SIRS, the median heart rates on postoperative day (POD) 2 were significantly lower in the sivelestat-treated group than in the control group (P= 0.007). The median arterial oxygen pressure/fraction of inspired oxygen ratio of the sivelestat-treated group were significantly higher than those of the control group on POD 1 and POD 7 (POD 1: 372.0 [range 284.0-475.0] vs 322.5 [243.5-380.0], respectively, P= 0.040; POD 7: 377.2 [339.5-430.0] vs 357.6 [240.0-392.8], P= 0.031). Postoperative white blood cell counts, serum C-reactive protein levels, plasma interleukin-1beta, tumor necrosis factor-alpha levels, and plasma NE levels did not differ significantly between the two groups at any point during the postoperative course, nor did serum Krebs von den Lungen 6, surfactant protein-A, or surfactant protein-D levels, which were used as markers of alveolar type II epithelial cells to evaluate the severity of lung injury. Plasma interleukin-8 levels were significantly lower in the sivelestat-treated group than in the control group on POD 3 (P= 0.040). In conclusion, perioperative administration of sivelestat sodium hydrate (starting at the beginning of surgery) mitigated postoperative hypoxia, partially suppressed postoperative hypercytokinemia, shortened the duration of SIRS, and stabilized postoperative circulatory status after thoracoscopic esophagectomy.

Successful long-term management of adenomyosis associated with deep thrombosis by low-dose gonadotropin-releasing hormone agonist therapy.

We report the case of a patient with adenomyosis complicated by deep vein thrombosis in whom low-dose gonadotropin-releasing hormone agonist (GnRHa) therapy was useful as a uterus-conserving therapeutic option. The patient was a 34-year-old nulliparous woman who presented with edema and pain in the left lower leg. The patient had been treated with four cycles of GnRHa therapy for adenomyosis and repeatedly experienced chronic pelvic pain, dysmenorrhea and anemia due to hypermenorrhea. Leg venography confirmed deep vein thrombosis, and thrombolytic therapy was performed to eliminate symptoms. Because the patient strongly wanted to conserve the uterus, low-dose GnRHa therapy was initiated. The patient is currently taking 450 microg/day buserelin acetate nasally (regular dose: 900 microg/day), and estradiol levels have been maintained at 24-50 pg/ml. Anemia, leg numbness and chronic pelvic pain have dissipated, and the patient has not experienced estrogen deficiency symptoms for more than two years.

Publisher Correction: Shedding new light on the Crab with polarized X-rays.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Von Willebrand Factor Deposition and ADAMTS-13 Consumption in Allograft Tissue of Thrombotic Microangiopathy-like Disorder After Living Donor Liver Transplantation: A Case Report.

BACKGROUND: Thrombotic microangiopathy (TMA) pathogenesis after living donor liver transplantation (LDLT) is thought to be caused by release of unusually large von Willebrand factor multimers (UL-vWFMs) resulting from sinusoidal endothelial cell damage and induction of platelet adhesion and aggregation. A decrease in a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13 (ADAMTS-13) that cleave UL-vWFMs might cause excessive UL-vWFMs activity and result in platelet thrombus formation. However, this phenomenon has not undergone a full pathologic assessment. PROCEDURES: A 60-year-old man was diagnosed with hepatitis C-related end-stage cirrhosis. His son was the donor, and he underwent LDLT. On postoperative day 44, his laboratory findings met most TMA diagnostic criteria, and he was diagnosed with TMA-like disorder (TMALD). Localization of CD42b as a platelet marker, vWF, and ADAMTS-13 in allograft tissue of this patient were evaluated using immunohistochemistry. RESULTS: CD42b expression was observed as platelet aggregates attached to hepatocytes or within the hepatocyte cytoplasm, a morphology called extravasated platelet aggregation (EPA). vWF expression was observed mainly as deposited compact clusters, and ADAMTS-13 expression resembled distinct dots throughout the liver tissue. CONCLUSION: These findings suggest that EPA indicated sinusoidal endothelial cell damage followed by detachment, and vWF deposition resulted from UL-vWFM oversynthesis. ADAMTS-13 might be consumed in the allograft tissue to cleave UL-vWFMs, but ADAMTS-13 levels might be insufficient to cleave all the deposited UL-vWFMs. We present the case of an LDLT recipient diagnosed with TMALD using blood tests, which showed the presence of TMA pathogenesis in the allograft.

Shedding new light on the Crab with polarized X-rays.

Strong magnetic fields, synchrotron emission, and Compton scattering are omnipresent in compact celestial X-ray sources. Emissions in the X-ray energy band are consequently expected to be linearly polarized. X-ray polarimetry provides a unique diagnostic to study the location and fundamental mechanisms behind emission processes. The polarization of emissions from a bright celestial X-ray source, the Crab, is reported here for the first time in the hard X-ray band (~20-160 keV). The Crab is a complex system consisting of a central pulsar, a diffuse pulsar wind nebula, as well as structures in the inner nebula including a jet and torus. Measurements are made by a purpose-built and calibrated polarimeter, PoGO+. The polarization vector is found to be aligned with the spin axis of the pulsar for a polarization fraction, PF = (20.9 ± 5.0)%. This is higher than that of the optical diffuse nebula, implying a more compact emission site, though not as compact as, e.g., the synchrotron knot. Contrary to measurements at higher energies, no significant temporal evolution of phase-integrated polarisation parameters is observed. The polarization parameters for the pulsar itself are measured for the first time in the X-ray energy band and are consistent with observations at optical wavelengths.

Magnetic field effects of photocarrier generation in bulk heterojunctions at low temperature.

We report an experimental investigation of the magnetic field effect (MFE) in polymer bulk heterojunction devices at temperatures below 10 K using photocarrier extraction by linearly increasing voltages. The examined devices were composed of an active layer of poly(3-hexylthiophene) and [6,6]-phenyl-C61-butyric acid methyl ester. In the experiments, the delay time (td) dependence of the MFE was investigated in detail. For td < 80 µs, a positive MFE was observed in the field region B < 0.1 T and a negative MFE was observed for B > 0.2 T. For td > 8 ms, only a positive MFE proportional to B2 was observed. For the photocurrent pulse detected immediately after light irradiation, the MFE was negligibly small. In a high magnetic field of 15 T, a significant MFE exceeding 80% was observed at 1.8 K for td = 800 ms. We discuss the results based on a model of triplet-singlet (or singlet-triplet) conversion in the magnetic field and estimate the exchange integral for the charge-transfer exciton in this photovoltaic cell.

Ischemia reperfusion-facilitated sinusoidal endothelial cell injury in liver transplantation and the resulting impact of extravasated platelet aggregation.

BACKGROUND: The exact sequence of events leading to ultimate hepatocellular damage following ischemia/reperfusion (I/R) is incompletely understood. In this article, we review a mechanism of organ dysfunction after hepatic I/R or immunosuppressive treatment, in addition to the potential of liver sinusoidal endothelial cell (LSEC) protection and antiplatelet treatment for the suppression of hepatocellular damage. METHODS: A review of the literature, utilizing PubMed-NCBI, was used to provide information on the components necessary for the development of hepatocellular damage following I/R. RESULTS: It is well-established that LSECs damage following hepatic I/R or immunosuppressive treatment followed by extravasated platelet aggregation (EPA) is the root cause of organ dysfunction in liver transplantation. We have classified three phases, from LSECs damage to organ dysfunction, utilizing the predicted pathogenic mechanism of sinusoidal obstruction syndrome. The first phase is detachment of LSECs and sinusoidal wall destruction after LSECs injury by hepatic I/R or immunosuppressive treatment. The second phase is EPA, accomplished by sinusoidal wall destruction. The various growth factors, including thromboxane A2, serotonin, transforming growth factor-beta and plasminogen activator inhibitor-1, released by EPA in the Disse's space of zone three, induce portal hypertension and the progression of hepatic fibrosis. The third phase is organ dysfunction following portal hypertension, hepatic fibrosis, and suppressed liver regeneration through various growth factors secreted by EPA. CONCLUSION: We suggest that EPA in the space of Disse, initiated by LSECs damage due to hepatic I/R or immunosuppressive treatment, and activated platelets may primarily contribute to liver damage in liver transplantation. Endothelial protective therapy or antiplatelet treatment may be useful in the treatment of hepatic I/R following EPA.

Hepatocyte growth factor transforms immortalized mouse liver epithelial cells.

Transforming activity of hepatocyte growth factor (HGF) was demonstrated utilizing immortalized but not fully transformed mouse hepatocytes (MLE-10). Rat HGF cDNA, expressed under the control of a cytomegalovirus promoter, was transfected together with the neomycin resistance gene (PSV2neo) into MLE-10 cells by the calcium phosphate method, and propagated G418-resistant colonies were harvested colony by colony. After checking for integration and expression of exogenous HGF, five cell lines (MLE-10-HGF-1-5) were established. Three cell lines transfected with the vector only (MLE-10-CMV-1-3) were also established in the same manner. All MLE-10-HGF cell lines grew much faster than the MLE-10-CMV and original MLE-10 cells in culture and produced large colonies in soft agar, which colony production was blocked by the addition of anti-HGF antibody to the agar. After addition of HGF, original and MLE-10-CMV lines produced colonies in soft agar. The high-HGF-production lines (MLE-10-HGF-4 and -5) also gave rise to tumors within 2 weeks when implanted into the nude mice subcutis. In contrast, all MLE-10-CMV and original MLE-10 cells were negative in these growth assays. A rough parallelism between the level of HGF expression and the growth rate in both soft agar and nude mice subcutis was evident among MLE-10-HGF cell lines. Those with higher HGF production tended to grow in a scattered fashion in culture. High-affinity HGF receptor, HGFR/met, was expressed in MLE-10 and all the derived cell lines. Since HGF and/or HGFR/met gene expression is seen in various tumors and the serum HGF level is elevated in patients with hepatic disease, the present results indicate a possible significance of HGF and its receptor system in carcinogenesis, most probably via autocrine and/or paracrine mechanisms.

Multiple domains interacting with Gs in the porcine calcitonin receptor.

The molecular basis for Gs activation by the calcitonin (CT) receptor was investigated. Based upon the analysis of conserved regions in G protein-coupled receptors, two nonoverlapping regions in the heptahelical porcine CT receptor (CTR) were selected as candidate Gs-interacting domains: the third intracellular loop residues 327-344 (KLKESQEAESHMYLKAVR, P3 region) and the C-tail residues 404-418 (KRQWNQYQAQRWAGR, P4 region). To assess their Gs-interacting function, we expressed these sequences in hybrid insulin-like growth factor II receptors in which the receptor native Gi-interacting domain was converted to CTR sequences. In COS cells transfected with either P3- or P4-substituted hybrid receptor, membrane adenylyl cyclase activity significantly increased. The up-regulated activity of cAMP was confirmed by measuring the transcriptional activity of the cAMP response element in cells expressing either hybrid receptor. A mutant CTR lacking the P4 region maintained positive cAMP response but with an attenuated maximal capacity to produce cAMP. In contrast, we could not assess the function of the P3 region using a conventional deletion method, as CT bound poorly to cells transfected with either of the two P3-deficient CTRs (one lacking the P3 region and the other lacking P3 but having the P3 sequence in reverse orientation). These data suggest that the third intracellular loop and the C-tail in CTR have domain-specific roles in Gs activation and that the hybrid receptor approach used here, combined with a conventional mutagenesis approach, is useful for intact cell analysis and functional dissection of G protein-coupled receptors.

Decreased release of lipoprotein lipase is associated with vascular endothelial damage in NIDDM patients with microalbuminuria.

OBJECTIVE: To explore mechanisms for hypertriglyceridemia in diabetic patients with microalbuminuria, we examined an association between heparin-releasable lipoprotein lipase (LPL) and the von Willebrand factor (vWF), based on the hypothesis that LPL bound to endothelium is decreased by generalized endothelial damage. RESEARCH DESIGN AND METHODS: A total of 37 NIDDM patients with microalbuminuria and 69 patients with normoalbuminuria were studied. Plasma LPL mass in post-heparin plasma and plasma vWF antigen were quantified by sandwich-enzyme immunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: The NIDDM patients with microalbuminuria had higher plasma triglyceride (TG) and lower HDL cholesterol concentrations compared with the patients with normoalbuminuria. Heparin-releasable LPL mass was significantly lower in the microalbuminuric than in the normoalbuminuric subjects. Plasma level of vWF, a marker for endothelial damage, was significantly increased in microalbuminuric subjects compared with their normoalbuminuric counterparts. The LPL mass was inversely correlated with plasma vWF level at a high correlation coefficient value. The LPL mass was inversely related to TG and positively to HDL cholesterol concentrations. CONCLUSIONS: These results suggest that widespread endothelial damage occurred in NIDDM patients with microalbuminuria, thereby LPL moiety bound to the endothelium is decreased, which results in an impaired catabolism of TG-rich lipoproteins.

Efficacy of conversion gastrectomy following docetaxel, cisplatin, and S-1 therapy in potentially resectable stage IV gastric cancer.

BACKGROUND: Recent advances in gastric cancer chemotherapy have made macroscopic complete resection possible in some patients with stage IV disease. METHODS: We retrospectively investigated the efficacy of multimodal therapy with combined docetaxel, cisplatin, and S-1 (DCS) and conversion gastrectomy in 57 patients with stage IV gastric cancer. RESULTS: Of the 57 patients, 15 patients were categorized into potentially resectable case, which is defined as patients with single incurable factor including the upper abdominal para-aortic lymph node metastasis (16a2b1 PAN metastasis) or fewer than three peripheral liver metastases. The other 42 were categorized as initially unresectable. All of patients underwent DCS therapy, and then 34 patients underwent conversion gastrectomy. The 3-year overall survival (OS) rate among the patients who underwent conversion gastrectomy was 50.1% with MST of 29.9 months. They had significantly longer OS than patients who underwent DCS therapy alone (p < 0.01). Univariate analysis among the patents with conversion gastrectomy identified 16a2b1PAN metastasis, peritoneal metastasis, potential resectable case, R0 resection as significant prognostic factors. A 3-year OS in potential resectable cases was 92.9%. Multivariate analysis identified potential resectability as the only independent prognostic factor contributing to OS (HR 0.133, 95%CI 0.024-0. 744, p = 0.021). In contrast, clinical response was selected as the only independent prognostic factor in the subgroup of initially unresectable cases (HR 0.354, 95%CI 0.151-0.783, p = 0.021). CONCLUSION: Patients with potentially resectable disease had a remarkably good prognosis among stage IV gastric cancer patients, and might be ideal candidates for conversion gastrectomy following DCS therapy.

Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines.

Hepatocyte growth factor (HGF) has potent mitogenic activity for mature hepatocytes and various normal epithelial cells. We now have evidence that HGF at 1-10 ng/ml, strongly inhibits the growth of HepG2 hepatocellular carcinoma cells, B6/F1 melanoma cells and KB squamous carcinoma cells. These tumor cells express high affinity receptors for HGF with a Kd of 25-28 pM, similar to findings with hepatocytes. HGF at 1-100 ng/ml had no significant cytolytic effect on tumor cells. Therefore, the anti-proliferative effect of HGF on tumor cells seems to be cytostatic, not cytolytic. As HGF apparently has bidirectional effects on cell growth, the possibility that it can serve as an anti-tumor agent merits attention.

Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors.

We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC50 of 0.15 micrograms/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount of DNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time.

Multipurpose robot for automated cycle sequencing.

Paramagnetic beads have the superior advantages of easy separation and resuspension by controlling the magnetic filed. Previously, we have developed Magtration technology to automate paramagnetic bead handling and have built several automated instruments that handle 1-12 samples simultaneously. To achieve more high-throughput sample processing, two types of a 96-arrayed Integrated Magtration Unit (IMU) were developed, one installed with electromagnets and the other with thin rod-shaped magnets made of neodymium. A multipurpose robot (SX-96GC) equipped with the IMU was also developed for fully automatic processing of 96 samples in parallel. The cleanup of dye-terminator sequencing products was performed using the robot installed with the permanent magnet version of IMU. The results had quality comparable to those by the same protocol in manual handling or to those by the conventional protocols. The robot processed 96 samples in a microplate within 30 min. The protocol that can purify 384 samples within 1 h by processing two microplates concurrently was successfully designed.

Inhibitory effect of a serine protease inhibitor, FOY-305 on the invasion and metastasis of human pancreatic cancers.

We examined the inhibitory effect of a serine protease inhibitor, FOY-305, on the invasion and metastasis of human pancreatic cancers. The in vitro matrigel invasion assay showed that the invasiveness of Capan-1 human pancreatic cancer cells was inhibited by FOY-305 treatment in a dose-dependent manner at concentrations greater than 100 nM. Intrasplenic injection of Capan-1 cells in nude mice resulted in frequent metastases to liver, however, its incidence was significantly decreased by FOY-305 treatment. These findings suggest that a serine protease inhibitor, FOY-305 can inhibit tumor invasion and metastasis by blocking the serine protease-mediated activation cascade.

Activated lansoprazole inhibits cancer cell adhesion to extracellular matrix components.

Integrins play an important role in tumor dissemination. The purpose of this study was to examine whether a SH-reactive reagent inhibits the adhesion of the human pancreatic cancer cell line AsPC-1 to extracellular matrix components. Activated lansoprazole (AG-2000) was used as the SH-reactive reagent because this compound is known to react with SH groups but does not permeate the cell membrane. The effect of AG-2000 on the adhesion of AsPC-1 cells to matrix was examined, using both an in vitro adhesion assay and an in vivo nude mouse xenograft model of peritoneal implantation. In the in vitro adhesion assay, a 60-min exposure of AsPC-1 cells to AG-2000 resulted in a dose-dependent inhibition of AsPC-1 cell adhesion to laminin, fibronectin and type IV collagen, although AG-2000 did not affect the viability of AsPC-1 cells by MTT assay. In the in vivo assessment of AsPC-1 cell implantation, the AsPC-1 cells were initially preincubated with AG-2000 for 60 min to ensure adequate exposure of the AsPC-1 cells to AG-2000 before intraperitoneal injection. AG-2000 significantly inhibited the peritoneal implantation of the AsPC-1 cells in nude mice. These findings suggest that a short exposure of cancer cells to AG-2000 can inhibit cancer cell adhesion to extracellular matrix components.