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Severe hypertriglyceridemia is a pathological condition caused by genetic factors alone or in combination with environmental factors, sometimes leading to acute pancreatitis (AP). In this study, exome sequencing and biochemical analyses were performed in 4 patients with hypertriglyceridemia complicated by obesity or diabetes with a history of AP or decreased post-heparin LPL mass. In a patient with a history of AP, SNP rs199953320 resulting in LMF1 nonsense mutation and APOE rs7412 causing apolipoprotein E2 were both found in heterozygous form. Three patients were homozygous for APOA5 rs2075291, and one was heterozygous. ELISA and Western blot analysis of the serum revealed the existence of apolipoprotein A-V in the lipoprotein-free fraction regardless of the presence or absence of rs2075291; furthermore, the molecular weight of apolipoprotein A-V was different depending on the class of lipoprotein or lipoprotein-free fraction. Lipidomics analysis showed increased serum levels of sphingomyelin and many classes of glycerophospholipid; however, when individual patients were compared, the degree of increase in each class of phospholipid among cases did not coincide with the increases seen in total cholesterol and triglycerides. Moreover, phosphatidylcholine, lysophosphatidylinositol, and sphingomyelin levels tended to be higher in patients who experienced AP than those who did not, suggesting that these phospholipids may contribute to the onset of AP. In summary, this study revealed a new disease-causing gene mutation in LMF1, confirmed an association between overlapping of multiple gene mutations and severe hypertriglyceridemia, and suggested that some classes of phospholipid may be involved in the pathogenesis of AP.

Background and Aim: Immune checkpoint inhibitors have significantly improved the clinical outcomes of many cancer types, but they induce a range of immune-related adverse events (irAEs). Although adrenal insufficiency (AI) is a rare irAE, it can lead to serious consequences. This study aimed to determine the clinical features of patients with advanced non-small-cell lung cancer (NSCLC) who developed AI following pembrolizumab treatment., Methods: We retrospectively reviewed and analyzed the clinical data of all patients with NSCLC treated with pembrolizumab at Juntendo University Hospital from February 2017 to December 2020. The diagnosis of AI was established based on the Endocrine Emergency Guidance for the acute management of endocrine complications of checkpoint inhibitor therapy in the UK and the clinical practice guidelines of the Japan Endocrine Society., Result: AI was clinically suspected in 59 out of 186 patients treated with pembrolizumab, and 10 (5.4%) cases were confirmed. The symptoms included hyponatremia (n = 9), fatigue (n = 8), and loss of appetite (n = 6). All patients had low adrenocorticotropic hormone (ACTH) levels, and five patients were diagnosed with isolated ACTH deficiency. All patients completely recovered with corticosteroid replacement. The median time to onset of AI was 8.0 (range 3.8-15.2) months. The median progression-free survival in these patients was 22.4 (95% confidence interval 11.2-not reached) months., Conclusion: The incidence of AI among patients treated with pembrolizumab is more frequent than previously reported. In addition, secondary AI, especially isolated ACTH deficiency, is a major form of AI induced by pembrolizumab., (© 2022 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Background: The combination of immune-checkpoint inhibitors with chemotherapy has become the standard of treatment for non-small cell lung cancer (NSCLC) patients. However, the association between therapeutic efficacy and the development of immune-related adverse events (irAEs) remains unclear in patients treated with combination therapy. We aimed to investigate the frequency of irAEs, and the association between therapeutic efficacy and the development of irAEs in patients with NSCLC., Materials and Methods: We retrospectively surveyed patients with chemo-naïve advanced NSCLC who received pembrolizumab plus platinum-based chemotherapy or pembrolizumab monotherapy at Juntendo University Hospital, Japan, between February 2017 and May 2021., Results: Among 148 patients (median [range] age, 68 (33-85) years; 107 men [72.3%] and 41 women [27.7%]), 74 each received pembrolizumab plus chemotherapy and pembrolizumab monotherapy. IrAEs were observed in 46 (62.2%) and 41 patients (55.4%) in the combination therapy and monotherapy group, respectively. Patients with irAEs showed significantly longer progression-free survival (PFS) than those without irAEs in the combination therapy group (8.9 vs. 5.7 months; Hazard Ratio [HR], 0.53; 95% CI, 0.29-0.98; P = 0.041) and monotherapy group (11.7 vs. 5.0 months; HR, 0.40; 95% CI, 0.22-0.70; P = 0.001). In the multivariable analysis, development of irAEs was positively associated with PFS in both the groups (HR, 0.48; 95% CI, 0.26-0.89; P = 0.019 and HR, 0.38; 95% CI, 0.21-0.68; P < 0.01). In the inverse probability of treatment weighting adjusted analysis, development of irAEs was significantly associated with combination therapy (OR, 0.56; 95% CI, 0.34-0.91; P = 0.019)., Conclusion: Our study demonstrated that the incidence of irAEs was associated with favorable efficacy in patients treated with pembrolizumab plus chemotherapy, as well as pembrolizumab monotherapy. Also, the addition of chemotherapy to pembrolizumab significantly increased the incidence of irAEs., (© 2022. The Author(s).)

Background: The prognosis of patients with NSCLC harboring oncogenic driver gene alterations, such as EGFR gene mutations or ALK fusion, has improved dramatically with the advent of corresponding molecularly targeted drugs. As patients were followed up for about five years in most clinical trials, the long-term outcomes beyond 5 years are unclear. The objectives of this study are to explore the clinical course beyond five years of chemotherapy initiation and to investigate factors that lead to long-term survival., Methods: One hundred and seventy-seven patients with advanced, EGFR-mutated or ALK-rearranged NSCLC who received their first chemotherapy between December 2008 and September 2015 were included. Kaplan Meier curves were drawn for the total cohort and according to subgroups of patients' characteristics., Results: Median OS in the total cohort was 40.6 months, the one-year survival rate was 89%, the three-year survival rate was 54%, and the five-year survival rate was 28%. Median OS was 36.9 months in EGFR-mutated patients and 55.4 months in ALK-rearranged patients. The OS curve seemed to plateau after 72 months, and most of the patients who were still alive after more than five years are on treatment. Female sex, age under 75 years, an ECOG PS of 0 to 1, ALK rearrangement, postoperative recurrence, and presence of brain metastasis were significantly associated with longer OS., Conclusions: A tail plateau was found in the survival curves of patients with advanced, EGFR-mutated and ALK-rearranged NSCLC, but most were on treatment, especially with EGFR-mutated NSCLC., (© 2022. The Author(s).)

Background: Regimens combining pemetrexed (PEM) and immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) or programmed death-ligand 1 (PD-L1) are widely used for the treatment of advanced non-squamous non-small-cell lung cancer (NSq-NSCLC). Recently, PEM was shown to induce immunogenic cell death (ICD) and to enhance immune-regulatory genes. Some patients demonstrate an extremely long-term response to PEM. It is possible that the continued response in these patients is dependent on not only the pharmacological induction of cytotoxic cell death but also antitumor immunity. However, factors that can predict outcomes associated with long-term PEM administration using blood test results have not yet been elucidated. We investigated the clinical characteristics and predictive factors in patients with advanced NSq-NSCLC who underwent long-term PEM maintenance therapy., Methods: In total, 504 patients with advanced NSq-NSCLC who received PEM combination therapy/monotherapy (n = 414) or paclitaxel (PTX) combination therapy (n = 90) between January 2010 and November 2019 were recruited; 381 patients were retained for the final analysis. Patients treated with PEM (n = 301) were divided into subgroups according to the total cycles of PEM (≥ 17 [n = 25] for the long-term administration group and ≤ 16 [n = 276] for the intermediate/short-term group) and compared with another population (n = 80) treated with PTX combination regimen. We investigated clinical features and predictive biomarkers, focusing on immune-regulatory factors, absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), and PD-1 and PD-L1 expression, to predict long-term response to PEM., Results: The long-term PEM administration group exhibited a higher ALC and a lower NLR than the shorter-term group did. Both these markers displayed greater association with progression-free survival and overall survival in the PEM combination therapy group than in the PTX combination therapy group. Increased PD-1 lymphocytes were associated with the long-term PEM response group, as PD-L1 expression in tumors was associated with a high incidence of immune-related adverse effects following ICI administration., Conclusions: ALC, NLR, and PD-1 expression are PEM-mediated predictive biomarkers that are indirectly related to tumor immunity and can provide useful predictive information on the long-term response to PEM in patients with NSq-NSCLC.

Background: There are few studies that directly compare the effects of osimertinib on patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) T790M mutation between the generation of prior EGFR tyrosine kinase inhibitors (TKIs)., Methods: We retrospectively reviewed clinical data from the medical records of consecutive patients with advanced NSCLC who had developed resistance to first- or second-generation EGFR-TKIs due to EGFR T790M mutation and were subsequently treated with osimertinib at Juntendo University Hospital. In patients with available tumor samples, target amplicon sequencing analyses were performed to explore the genetic biomarkers., Results: A total of 38 patients with NSCLC harboring EGFR T790M mutation were treated with osimertinib. Eight patients were classified into group A (afatinib followed by osimertinib) and 30 patients were classified into group B (first-generation EGFR-TKI followed by osimertinib). Progression-free survival (PFS) was significantly longer in group A than in group B (median PFS; not reached vs. 11.0 months, P = 0.018). Fourteen patients had available tissue samples collected before osimertinib treatment for target sequencing. In group A we found no additional mutations, other than EGFR T790M mutation. On the other hand, there were three samples in which other mutations emerged, in addition to EGFR T790M mutation, in group B., Conclusions: PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR-TKIs. Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment., Key Points: Significant findings of the study: PFS of osimertinib was significantly longer in patients with NSCLC harboring EGFR T790M mutation after treatment with afatinib than in patients after treatment with first generation EGFR-TKIs., What This Study Adds: Additional mutations other than EGFR T790M may affect the efficacy of osimertinib treatment., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Aim: To evaluate the cytotoxicity and inflammatory response of different types of provisional restorative materials to mice gingival fibroblasts., Methods: Cytotoxicity of provisional material discs (thermal-polymerized acrylic resin; auto-polymerized acrylic resin; bisacrylic resin; nano-ceramic resin for CAD/CAM and prefabricated polymer block for CAD/CAM) to Mice (Balb/c) gingival cell were investigated under direct and indirect contact (extracts) at 24, 48 and 72 h, using the MTT and Alamar blue assays. Materials extracts (24 h) were applied to the cell culture (indirect contact) or cells were seeded on discs of provisional materials, and the cytotoxicity and production of IL-6, IL-1β and TNF-α after 24, 48 and 72 h were analyzed through MTT, Alamar Blue® and ELISA. Culture medium was used as control for indirect contact assay (extract) and the surfaces of the wells without discs of provisional materials were used as control for direct contact assay. Results were analysed statistically by ANOVA followed by the Bonferroni-Test correction. Statistically significant differences were considered if P was < .05., Results: Auto-polymerized and bisacrylic resins (direct contact) reduced cell viability after 24, 48 and 72 h compared to control (P < .05). Indirect contact (extract) was not cytotoxic to cells at all periods compared to control (P > .05). Auto-polymerized and bisacrylic resins increased IL-6, IL-1β and TNF-α levels mainly at 24 h when compared to the other materials (P < .001)., Conclusion: Auto-polymerized and bisacrylic were more cytotoxic to mice gingival fibroblasts. CAD/CAM nano ceramic resin and prefabricated polymer blocks are more predictable materials to preserve the periodontal soft tissues., Competing Interests: Declaration of Competing Interest Manuscript title “Cytotoxicity and inflammatory response of different types of provisional restorative materials” The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Interstitial lung diseases induced by anticancer agents (ILD-AA) are rare adverse effects of anticancer therapy. However, prognostic biomarkers for ILD-AA have not been identified in patients with advanced lung cancer. Our aim was to analyze the association between serum biomarkers sialylated carbohydrate antigen Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), and clinical characteristics in patients diagnosed with ILD-AA., Methods: Between April 2011 and March 2016, 1224 advanced lung cancer patients received cytotoxic agents and epidermal growth factor receptor tyrosine kinase inhibitors at Juntendo University Hospital and Juntendo University Urayasu Hospital. Of these patients, those diagnosed with ILD-AA were enrolled in this case control study. ΔKL-6 and ΔSP-D were defined as the difference between the levels at the onset of ILD-AA and their respective levels prior to development of ILD-AA. We evaluated KL-6 and SP-D at the onset of ILD-AA, ΔKL-6 and ΔSP-D, the risk factors for death related to ILD-AA, the chest high resolution computed tomography (HRCT) findings, and survival time in patients diagnosed with ILD-AA., Results: Thirty-six patients diagnosed with ILD-AA were enrolled in this study. Among them, 14 patients died of ILD-AA. ΔSP-D in the patients who died was significantly higher than that in the patients who survived. However, ΔKL-6 did not differ significantly between the two groups. Moreover, ΔSP-D in patients who exhibited diffuse alveolar damage was significantly higher than that in the other patterns on HRCT. Receiver operating characteristic curve analysis was used to set the optimal cut off value for ΔSP-D at 398 ng/mL. Survival time for patients with high ΔSP-D (≥ 398 ng/mL) was significantly shorter than that for patients with low ΔSP-D. Multivariate analysis revealed that ΔSP-D was a significant prognostic factor of ILD-AA., Conclusions: This is the first research to evaluate high ΔSP-D (≥ 398 ng/mL) in patients with ILD-AA and to determine the risk factors for ILD-AA in advanced lung cancer patients. ΔSP-D might be a serum prognostic biomarker of ILD-AA. Clinicians should evaluate serum SP-D during chemotherapy and should carefully monitor the clinical course in patients with high ΔSP-D.

Background: Advanced non-small cell lung cancer (NSCLC) patients often develop thromboembolic events, including cerebral infarction (CI). However, the relationship between advanced NSCLC and CI has not been thoroughly investigated. We examined the association between advanced NSCLC and CI and risk factors for CI in advanced or post-operative recurrent NSCLC patients., Methods: We retrospectively investigated 515 patients diagnosed with advanced or post-operative recurrent NSCLC at Juntendo University Hospital between April 2009 and March 2014., Results: Among the 515 patients evaluated, 15 patients (2.9%) developed CI after diagnosis of advanced or post-operative recurrent NSCLC. Univariate and multivariate analyses were conducted, and brain metastasis was the only significant independent risk factor for CI (odds ratio 5.24, 95% confidence interval 1.72-16.10, p = 0.004). The incidence was 6.3% in these patients. The median survival time was 36 days, and 1-year survival rate was 6.7% after development of CI. Overall survival from diagnosis of advanced NSCLC or post-operative recurrence was significantly shorter in patients with CI than in patients without CI (223 days versus 895 days; HR, 3.46; 95% confidence interval, 2.04-6.02; p = 0.001)., Conclusions: The incidence of CI is high in advanced or post-operative recurrent NSCLC, and is especially higher in patients with brain metastasis than in those without brain metastasis. Moreover, CI may affect patient's prognosis. Careful monitoring for the development of CI in patients with advanced or post-operative recurrent NSCLC is needed, especially for patients with brain metastasis.

Photoelastic analysis was used to evaluate the biomechanical behaviour of implant-supported, double-screwed crowns with different connection systems and cantilever lengths. Three models were made in PL-2 photoelastic resin and divided into six groups, on the basis of the implant connection system (external hexagon [EH] or Morse taper [MT]), type of abutment (Mini Pilar [Neodent, Curitiba, Paraná, Brazil] or "UCLA") and number of crowns in the cantilever (one or two). The implant-prosthesis unit was placed in a circular polariscope. Occlusal surfaces of the crowns were subjected to 100-N loads in the axial and oblique (45°) directions in a universal testing machine (EMIC). Generated stresses were recorded and analysed qualitatively in a graphics program (Adobe Photoshop). Under axial loading, all of the groups had similar numbers of fringes, which were increased when the crowns were subjected to oblique loading. The highest number of fringes was found during oblique loading in the EH + Mini Pilar group. In conclusion, although the type of implant connection system did not have a direct influence on the stress distribution for axial loading, the cantilever length did have a direct influence on stress distribution. Models with two crowns in the cantilever showed more stress, with a greater concentration of force on the cervical part of the implant.

Purpose: Docetaxel or pemetrexed monotherapy is recommended either as a second-line treatment for non-small cell lung cancer (NSCLC) patients without EGFR mutation or ALK fusion genes or as a third-line treatment for patients with EGFR mutation or ALK fusion. However, efficacy and survival for these two settings have not been compared, leaving it unclear whether these two populations can be included in the same clinical trials. Moreover, prognostic factors for patients who are recommended to receive docetaxel/pemetrexed monotherapy are largely unknown., Methods: Docetaxel or pemetrexed was administered to 67 EGFR wild-type patients as a second-line treatment following one platinum-based combination chemotherapy and to 17 EGFR mutant patients as a third-line treatment following EGFR tyrosine kinase inhibitors and one platinum-based combination chemotherapy. Docetaxel and pemetrexed were administered at 60 and 500 mg/m(2), respectively, every 3 weeks until disease progression, intolerable toxicity, or patient refusal. Overall survivals (OSs) between the two groups were compared using the log-rank test, and prognostic factors were evaluated via Cox's proportional hazards models., Results: The median OS was 345.5 days in the EGFR wild-type second-line group and 616 days in the EGFR mutant third-line group. Multivariate analyses revealed that the stage before first-line treatment, performance status, and EGFR status were significant prognostic factors., Conclusions: When planning clinical studies of NSCLC patients recommended to receive docetaxel or pemetrexed as single-agent chemotherapy, the EGFR status and stage before first-line treatment should be considered as stratification factors of randomized clinical studies.

Background: Non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD) need to be approached carefully given the high incidence of pulmonary toxicity. Pemetrexed (PEM) is the key drug for the treatment of NSCLC. However, its safety, especially with respect to the exacerbation of ILD, and efficacy in NSCLC patients with ILD have yet to be established., Method: We investigated the safety and efficacy of PEM monotherapy in NSCLC patients with or without idiopathic interstitial pneumonia (IIPs). The medical charts of these patients were retrospectively reviewed., Results: Twenty-five patients diagnosed as having IIPs (IIPs group) and 88 patients without ILD (non-ILD group) were treated with PEM monotherapy at Juntendo University Hospital between 2009 and 2013. In the IIPs group, 12 patients were found to have usual interstitial pneumonitis (UIP) on chest computed tomography (CT) (UIP group) and the other 13 patients showed a non-UIP pattern on chest CT (non-UIP IIPs group). Three patients in the IIPs group (2 in the UIP group and 1 in the non-UIP IIPs group) and 1 in the non-ILD group developed pulmonary toxicity during treatment (3.5% overall, 12.0% in the IIPs group versus 1.1% in the non-ILD group). Moreover, all 3 patients in the IIPs group died of pulmonary toxicity. Overall survival tended to be longer in the non-ILD group than in the IIPs group (p = 0.08). Multivariate analyses demonstrated that IIPs was the only significant independent risk factor for PEM-related pulmonary toxicity., Conclusion: We found that the incidence of PEM-related pulmonary toxicity was significantly higher amongst NSCLC patients with IIPs than among those without IIPs. Particular care must be taken when administering PEM to treat NSCLC patients with IIPs.

Background: Since advanced non-small cell lung cancer (NSCLC) patients with the interstitial lung disease (ILD) have been excluded from clinical trials, it is uncertain whether chemotherapy really provides a benefit to these patients., Patients and Methods: Fifteen advanced NSCLC patients with ILD that was detected on the chest X-rays were enrolled in this study. Carboplatin plus paclitaxel was administered by two methods (method A or method B). Method A: Carboplatin (AUC 6, day 1) and paclitaxel (70 mg/m(2), days 1, 8, 15) were administered every four weeks. Method B: Carboplatin (AUC 2, day 1, 8, 15) and paclitaxel (60 mg/m(2), days 1, 8, 15) were administered every four weeks., Results: The response rate and the disease control rate were 33% and 53%. The median progression-free survival and the median overall survival time were 2.5 months and 7.0 months, respectively. The hematological toxicities were tolerable, but a grade 3 or higher pneumonitis was observed in 4 patients (27%)., Conclusion: Carboplatin plus weekly paclitaxel must be administered carefully to advanced NSCLC patients with ILD that is detected on chest X-rays after a sufficient evaluation of the risks and the benefits.

Purpose: The purpose of this study was to compare the artificial tooth positional changes following the flasking and polymerization of complete dentures by a combination of two flasking methods and two polymerization techniques using computer graphic measurements., Materials and Methods: Four groups of waxed complete dentures (n = 10) were invested and polymerized using the following techniques: (1) adding a second investment layer of gypsum and conventional water bath polymerization (Control), (2) adding a second investment layer of gypsum and polymerization with microwave energy (Gypmicro), (3) adding a second investment layer of silicone (Zetalabor) and conventional polymerization (Silwater), and (4) adding a second investment layer of silicone and polymerization with microwave energy (Silmicro). For each specimen, six segments of interdental distances (A to F) were measured to determine the artificial tooth positions in the waxed and polymerized stages using software program AutoCad R14. The mean values of the changes were statistically compared by univariate ANOVA with Tukey post-hoc test at 5% significance., Results: There were no significant differences among the four groups, except for segment D of the Silmicro group (-0.004 +/- 0.032 cm) in relation to the Gypwater group (0.044 +/- 0.031 cm) (p < 0.05), which presented, respectively, expansion and shrinkage after polymerization., Conclusions: Within the limitations of this study, it was concluded that although the differences were not statistically significant, the use of a silicone investment layer when flasking complete dentures resulted in the least positional changes of the artificial teeth regardless of the polymerization technique.

Purpose: To investigate the physical imaging properties and detection of simulated microcalcifications of a new computed radiography (CR) system with a pixel size of 50 microm for digital mammography., Materials and Methods: New and conventional CR were employed in this study. The new CR system included a high-resolution imaging plate coupled with the FCR5000MA (50 microm pixel pitch) including transparent support and a dual-sided reader. The conventional CR system was coupled with the FCR9000 (100 microm pixel pitch). Modulation transfer functions (MTFs) and Wiener spectra (WS) of the new and conventional CR systems were measured. Observer performance tests were conducted to compare the effects of pixel size (50 microm vs. 100 microm) on the diagnostic accuracy of CR systems in the detection of simulated microcalcifications., Results: The presampling MTF of the new CR system was higher at high frequencies than the conventional CR system. The WS of the new CR system was comparable to that of the conventional CR system at all frequencies. The area under the receiver operating characteristic (ROC) curve (Az) obtained with the new CR and the conventional CR systems were 0.84 and 0.79, respectively. Results showed that the detection of simulated clustered microcalcifications was significantly improved by use of the new CR system compared with the conventional CR system (p<0.05)., Conclusion: The new CR mammography system improved physical imaging properties and detection of simulated microcalcifications over conventional CR mammography.

We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ-S1 cells, inhibits the serum-induced motility of FBJ-LL cells and that the metastatic potential of FBJ-LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999;83:685-91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ-LL cell motility. In the present study, the HGF-induced motility of FBJ-S1 cells was found to be one-thirtieth that of FBJ-LL cells. This motility of GD1a-expressing transfectants, which were produced by transfection of FBJ-LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a-pretreated FBJ-LL cells, indicating that GD1a inhibits the HGF-induced motility of FBJ-LL cells. The expression of the HGF receptor c-Met on FBJ-S1 cells, FBJ-LL cells, transfectants and a mock-transfectant was almost the same. The level of tyrosine phosphorylation of c-Met after HGF stimulation in FBJ-S1 cells, GD1a-pretreated FBJ-LL cells and a GD1a-expressing transfectant was significantly lower than in FBJ-LL cells and a mock-transfectant. These findings suggested that GD1a inhibits the HGF-induced motility of FBJ-LL cells through suppression of tyrosine phosphorylation of c-Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c-Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c-Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF-induced autophosphorylation of c-Met was suppressed. These results suggest that GD1a acts as a negative regulator of c-Met in cancer cells., (Copyright 2001 Wiley-Liss, Inc.)

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).

Modification of proteins by long-term incubation with glucose leads to the formation of advanced glycation end products (AGE). Recent immunological demonstration of the presence of AGE proteins in several human tissues suggests that they may be involved in aging, diabetic complications and atherosclerosis. AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macrophage scavenger receptor (MSR). In the present study, we examined whether MSR could mediate the endocytic uptake of AGE proteins by using Chinese hamster ovary (CHO) cells overexpressing bovine type II MSR (CHO-SRII). 125I-labelled AGE bovine serum albumin (125I-AGE-BSA) as well as 125I-acetylated low-density lipoprotein (125I-acetyl-LDL) underwent endocytic degradation by CHO-SRII cells, but not by control CHO cells. Endocytic degradation of 125I-acetyl-LDL and 125I-AGE-BSA by CHO-SRII cells was significantly inhibited by unlabeled AGE-BSA, as well as by acetyl-LDL. Immunoelectron microscopic studies using both AGE-BSA conjugated with gold particles and anti-(bovine MSR) antibody (D2) revealed co-localization of gold particles and the reactive sites for the antibody at coated pits of plasma membranes as well as in endosomes. These results clearly show that MSR mediates the endocytic uptake and degradation of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.

We examined the structural changes in relaxed glycerinated rabbit psoas muscle fibers induced by modification of the myosin heads with p-phenylenedimaleimide (p-PDM), which reacts with sulfhydryls on the myosin head to cause its loss of ability to combine with actin and to hydrolyse ATP. In the longitudinal sections of both chemically fixed and quickly frozen muscle fibers, ladder-like structures, interpreted as the myosin heads extending nearly at right angles with the thick filaments, were more prominent in the p-PDM-modified fibers than in the control fibers. Fourier transform diffractograms of the longitudinal sections exhibited a distinct 14.3-nm meridional reflection, which arises from axial spacing of the myosin heads on the thick filament, in the p-PDM-modified fibers, but not in the control fibers. These results indicate that the p-PDM-modification of the myosin heads causes an increase in the regularity of myosin head arrangement on the thick filament.

Metabolic pathways of cyanate in rats were studied by means of measurements of cyanate, carbamyl phosphate and S-carbamyl group. Approximately 30-50% of cyanate administered to rats (0.5 mmol/kg body weight) was found in buffered gastric contents, and was also detected as ammonia liberated by acid hydrolysis. However, the gastric excretion of cyanate was a temporary phenomenon just after cyanate administration. Biliary and urinary excretion of cyanate and acid-soluble S-carbamyl group are minor metabolic pathways.

Mechanisms of ventricular tachycardia induced by local application of a properly timed premature stimulus were studied with routine microelectrode technique and extracellular recordings on a ventricular sheet. Thinly sliced preparations obtained from subepicardial muscle of the canine ventricle were used as an approximation of a two-dimensional model. On these preparations, spontaneously sustained tachycardia easily induced by a single premature stimulus. Since delayed after-depolarizations were never evoked by frequent stimulations even in the K+-free and high-Ca++ media, these tachycardias seemed to be induced by re-entrant and circus movement mechanisms. To analyse the re-entrant mechanisms, action potentials generated by normal driving stimuli were recorded from multiple points (40 approximately 50 points) and the spreads of the depolarization and repolarization phases of the action potentials were mapped. The depolarizing wave front on the map always showed a circular or elliptical pattern. Whenever the pattern of spread of the repolarizing wave was similar to that of the depolarizing wave, sustained tachycardia was never brought about by any premature stimulus. On the other hand, when the map of the spread of the repolarizing wave was very complicated and mixed with that of the depolarizing wave, sustained tachycardia was frequently induced. From the above results, it is suggested that the nonuniform recovery of excitability plays a role in the generation of sustained tachycardia. Moreover, a portion of the unidirectional block of the premature impulse was determined by calculated using the conduction velocity of the premature impulse and the effective refractory period in each cell; then a route of re-entry for the premature impulse was simulated.(ABSTRACT TRUNCATED AT 250 WORDS)