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Abstract: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Introduction: Approximately 10% of mutations in the EGFR gene in NSCLC are in-frame insertions in exon 20 (X20ins). These tumors usually do not respond to conventional EGFR tyrosine kinase inhibitors (TKIs). Several novel EGFR TKIs active for X20ins are in clinical development, including mobocertinib, which was recently approved by the U.S. Food and Drug Administration. However, acquired resistance during treatment with these TKIs still occurs as in the case of EGFR TKIs of earlier generations., Methods: We chronically exposed murine pro-B-cell line cells transduced with the five most common X20ins (A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, H773_V774insNPH and H773_V774insH) to mobocertinib in the presence of N-ethyl-N-nitrosourea and searched for secondary EGFR mutations. We evaluated the efficacies of several EGFR X20ins inhibitors, including zipalertinib and sunvozertinib, against cells with acquired resistant mutations., Results: All secondary mutations resulting in acquired resistance to mobocertinib were exclusively C797S in insFQEA and insSVD. However, in the case of other X20ins (insASV, insNPH, and insH), T790M or C797S secondary mutations contributed to acquired resistance to mobocertinib. The emergence of T790M was more frequent in cells treated with lower drug concentrations. Sunvozertinib exhibited good activity against resistant cells with T790M. Cells with C797S were refractory to all EGFR TKIs, except for erlotinib, which was active for insFQEA with C797S., Conclusions: T790M or C797S, depending on the original X20ins mutations, conferred acquired resistance to mobocertinib. Sunvozertinib may be the treatment of choice for patients with tumors resistant to mobocertinib because of T790M., (Copyright © 2023 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/μL, respectively. The detection limit of the MRSA bacterial burden using this system was 10 4 and 10 3  CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles., Competing Interests: Declaration of competing interest This study was funded by Kyorin Pharmaceutical Co., Ltd. The sponsor provided technical support for the GeneSoC® system examination, including system maintenance, during the study period. The sponsor had no control over the interpretation, writing, or publication of this work., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The differential diagnosis of a solitary solid-type lung nodule is diverse. 18F-fluorodeoxyglucose positron emission tomography (PET) has a high sensitivity in the diagnosis of solid-type lung cancers; however, PET-negative, solid-type lung cancers are rarely observed. In this study, we analyzed the clinical/genetic features and prognosis of PET-negative, solid-type lung cancers., Patients and Methods: Between January 2007 and February 2020, 709 patients with solid-type lung cancers (tumor size ≥2.0 cm) underwent pulmonary resection. Clinical, genetic, and prognostic features were evaluated in 27 patients (3.8%) with tumors showing negative PET results defined as SUVmax <2.0., Results: All 27 patients had lung adenocarcinoma; 23 had invasive adenocarcinomas and 4 had invasive mucinous adenocarcinomas. The PET-negative group showed high frequencies of females and never-smokers. Recurrence-free survival was significantly better in the PET-negative group compared with PET-positive counterparts extracted using propensity score matching from patients who underwent pulmonary resection during the same period (P = .0052). Furthermore, 83% of PET-negative, solid-type invasive lung adenocarcinoma patients harbored EGFR mutation, which was significantly higher than that of PET-positive, solid-type invasive lung adenocarcinoma patients (38%, n = 225) who received EGFR mutation testing in our cohort (P < .0001). PET-negative, solid-type lung adenocarcinoma patients with EGFR mutations had significantly better recurrence-free survival compared with PET-positive, solid-type lung adenocarcinoma patients with EGFR mutations extracted using propensity score matching (P = .0030)., Conclusion: PET-negative, solid-type lung cancers are characterized with a high incidence of EGFR mutation and a better prognosis compared with PET-positive, solid-type lung cancer., Competing Interests: Disclosure Dr. Suda has received research funding from Rain Therapeutics and Boehringer Ingelheim, has received honoraria from Boehringer Ingelheim, Chugai Pharmaceuticals, and AstraZeneca, and has been on the advisory board of AstraZeneca outside of the submitted work. Dr. Fujino has received research funding from Apollomics and an honorarium from Novartis outside of the submitted work. Dr. Hamada has received lecture fees from AstraZeneca and Chugai Pharmaceuticals outside of the submitted work. Dr. Chiba has received honorarium from Ethicon, Olympus, B-Braun, and Covidien outside of the submitted work. Dr. Mitsudomi has received research funding from Boehringer Ingelheim, AstraZeneca, Taiho, Ono Pharmaceuticals, Merck Sharp & Dohme, Eli Lilly, and Chugai Pharmaceuticals; has received lecture fees from AstraZeneca, Boehringer Ingelheim, Chugai Pharmaceuticals, Pfizer, Bristol-Myers Squibb, Eli Lilly, Merck Sharp & Dohme, Novartis, Merck Biopharma, Ono Pharmaceuticals, and Takeda Pharmaceuticals; and has been on the advisory board of AstraZeneca, Amgen, Janssen Pharma, Merck Sharp & Dohme, Novartis, and Puma Biotech outside of the submitted work. The remaining authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier Inc.)

Introduction: KRAS mutations have been recognized as undruggable for many years. Recently, novel KRAS G12C inhibitors, such as sotorasib and adagrasib, are being developed in clinical trials and have revealed promising results in metastatic NSCLC. Nevertheless, it is strongly anticipated that acquired resistance will limit their clinical use. In this study, we developed in vitro models of the KRAS G12C cancer, derived from resistant clones against sotorasib and adagrasib, and searched for secondary KRAS mutations as on-target resistance mechanisms to develop possible strategies to overcome such resistance., Methods: We chronically exposed Ba/F3 cells transduced with KRAS G12C to sotorasib or adagrasib in the presence of N-ethyl-N-nitrosourea and searched for secondary KRAS mutations. Strategies to overcome resistance were also investigated., Results: We generated 142 Ba/F3 clones resistant to either sotorasib or adagrasib, of which 124 (87%) harbored secondary KRAS mutations. There were 12 different secondary KRAS mutations. Y96D and Y96S were resistant to both inhibitors. A combination of novel SOS1 inhibitor, BI-3406, and trametinib had potent activity against this resistance. Although G13D, R68M, A59S and A59T, which were highly resistant to sotorasib, remained sensitive to adagrasib, Q99L was resistant to adagrasib but sensitive to sotorasib., Conclusions: We identified many secondary KRAS mutations causing resistance to sotorasib, adagrasib, or both, in vitro. The differential activities of these two inhibitors depending on the secondary mutations suggest sequential use in some cases. In addition, switching to BI-3406 plus trametinib might be a useful strategy to overcome acquired resistance owing to the secondary Y96D and Y96S mutations., (Copyright © 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Objective: Terminally ill patients with hematological malignancy tend to be treated aggressively. We aimed to clarify the status and costs of antimicrobial treatment of patients dying with hematological malignancies., Methods: This retrospective study was conducted in a Japanese acute hospital between September 2010 and August 2015. A total of 141 patients who stayed for 14 days or longer and died in the hospital were investigated., Results: The median patient age was 67 years (range, 22-93). Most patients were treated with antibacterial, antifungal, and antiviral agents (98%, 75%, and 27% of the patients, respectively) in the last 14 days of their lives. The frequency of antibiotics used in the last 7 days did not differ from that of the week before. The median cost of antimicrobials was 245,000 JPY (2227 USD), which reflected 16% of the total medical costs spent over the last 14 days. A subgroup analysis of the patients according to care policy (aggressive care policy (A) and palliative care policy (P), respectively) showed that the total medical cost in group P in the last 7 days decreased from that of the preceding week; however, the cost of antimicrobials did not lessen even in the last 7 days., Conclusions: Most patients dying with hematological malignancy were treated with a broad spectrum of antimicrobials. It appeared to be difficult to reduce, let alone discontinue antimicrobial treatment even in patients treated according to the palliative care policy. The optimal use of antibiotics for hematological patients in their end-of-life should be discussed., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Ultrasound-targeted microbubble destruction (UTMD) induces cellular inflow of drugs at low intensity, while high intensity eradicates tumor vessels. Since vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), VEGFR2-targeted microbubble (MB) might additionally increase the tissue specificity of drugs and thus improve antitumor effects. In addition, fixing the dual pulse intensity could maximize MB properties. This study evaluated the one-off (experiment 1) and cumulative (experiment 2) treatment effect of UTMD by regulating the dual pulse output applied to PDAC using VEGFR2-targeted MB., Methods: C57BL/6 mice inoculated with Pan-02 cells were allocated to five groups: VEGFR2-targeted MB+ gemcitabine (GEM), VEGFR2-targeted MB, non-targeted MB+GEM, GEM, and control groups. After injection of GEM or GEM and either VEGFR2-targeted or non-targeted MB, UTMD was applied for several minutes at low intensity followed by high intensity application. In experiment 1, mice were treated by the protocol described above and then euthanized immediately or at the tumor diameter doubling time (TDT). In experiment 2, the same protocol was repeated weekly and mice were euthanized at TDT regardless of protocol completion. Histological analysis by CD31 and VEGFR2 staining provided microvascular density (MVD) and VEGFR2 expression along vessels (VEGFR2v) or intra/peripheral cells (VEGFR2c)., Results: In experiment 1, TDT was significantly longer in the VEGFR2-targeted MB+GEM group compared to the non-targeted MB+GEM, GEM, and control groups, while the VEGFR2-targeted MB group showed no statistical significance. MVD and VEGFR2v in the immediate euthanasia was significantly lower in the VEGFR2-targeted MB+GEM and VEGFR2-targeted MB groups than other conditions. In experiment 2, the VEGFR2-targeted MB+GEM group produced significantly longer TDT than the GEM or control groups, whereas the VEGFR2-targeted MB group showed no significant difference. Histology revealed significantly reduced VEGFR2v and VEGFR2c in the VEGFR2-targeted and non-targeted MB+GEM groups, while only VEGFR2v was significantly less in the VEGFR2-targeted MB group., Conclusions: UTMD-mediated GEM therapy with the dual pulse application using VEGFR2-targeted MB substantially suppresses PDCA growth., (Copyright © 2020. Published by Elsevier B.V.)

Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with β-TRCP, a substrate receptor subunit of the SCF β - TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. β-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that β-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: MNNG HOS transforming gene (MET) exon 14 mutations in lung cancer, including exon 14 skipping and point mutations, have been attracting the attention of thoracic oncologists as new therapeutic targets. Tumors with these mutations almost always acquire resistance, which also occurs in other oncogene-addicted lung cancers. However, the resistance mechanisms and treatment strategies are not fully understood., Methods: We generated Ba/F3 cells expressing MET exon 14 mutations by retroviral gene transfer. The sensitivities of these cells to eight MET-tyrosine kinase inhibitors (TKIs) were determined using a colorimetric assay. In addition, using N-ethyl-N-nitrosourea mutagenesis, we generated resistant clones, searched for secondary MET mutations, and then examined the sensitivities of these resistant cells to different TKIs., Results: Ba/F3 cells transfected with MET mutations grew in the absence of interleukin-3, indicating their oncogenic activity. These cells were sensitive to all MET-TKIs except tivantinib. We identified a variety of secondary mutations. D1228 and Y1230 were common sites for resistance mutations for type I TKIs, which bind the active form of MET, whereas L1195 and F1200 were common sites for type II TKIs, which bind the inactive form. In general, resistance mutations against type I were sensitive to type II, and vice versa., Conclusions: MET-TKIs inhibited the growth of cells with MET exon 14 mutations. We also identified mutation sites specific for TKI types as resistance mechanisms and complementary activities between type I and type II inhibitors against those mutations. These finding should provide relevant clinical implication for treating patients with lung cancer harboring MET exon 14 mutations., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Introduction: Dacomitinib was superior to gefitinib in terms of progression-free survival in patients with EGFR-mutant lung cancer in a recent ARCHER 1050 trial. However, despite a marked initial response, lung cancers eventually acquire resistance to these inhibitors. This study aimed to elucidate the mechanisms of acquired resistance to dacomitinib in vitro., Methods: Dacomitinib-resistant clones were established by exposure to fixed concentrations of dacomitinib by using N-ethyl-N-nitrosourea (ENU) mutagenesis or by chronic exposure to increasing concentrations of dacomitinib without ENU. EGFR secondary mutations were analyzed by Sanger sequencing. Time to resistance in each clone was compared according to the mutational status. EGFR Del19, L858R, and G719A mutations were introduced into Ba/F3 cells by using retroviral vectors., Results: Chronic exposure to dacomitinib without ENU induced T790M in Ba/F3 cells expressing Del19. ENU mutagenesis resulted in 171 dacomitinib-resistant clones. Among these clones, 90% acquired T790M. However, C797S occurred in 11% of L858R-mutant clones (four of 35) and in 24% of G719A-mutant clones (12 of 38) established by using low-dose dacomitinib. Time to resistance was not significantly different between T790M- and C797S-mutant clones in both of L858R clones (p = 0.93) and G719A clones (p = 0.86). Cells expressing Del19 that acquired T790M were sensitive to osimertinib, whereas cells with L858R plus C797S mutations were sensitive to gefitinib or erlotinib., Conclusions: These in vitro data demonstrate that dacomitinib can directly induce T790M or C797S secondary mutations. Our data suggest the importance of analyzing these secondary mutations because appropriate selection of EGFR inhibitors could overcome acquired resistance to dacomitinib in a subset of lung cancers., (Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Chromium speciation by spectrophotometric determination of hexavalent chromium (Cr(VI)) with diphenylcarbazide (DPC) has several problems. These include: (1) the inability to directly detect trivalent chromium (Cr(III)) with DPC, (2) positive interference in Cr(VI) determination by other metal cations and (3) negative interference by any reducing agent present in the sample. These are addressed with an ion transfer device (ITD) in a flow injection analysis system. We previously developed the ITD for electrodialytic separations. Here we separate oppositely charged Cr(III) and Cr(VI) species by the ITD into two different acceptor solutions within ~5 s. The acceptor solutions consist of buffered H 2 O 2 to oxidize the Cr(III) to Cr(VI). Then DPC is added to either acceptor to measure Cr(III) and Cr(VI) spectrophotometrically. The system was optimized to provide the same response for Cr(VI) and Cr(III) with limits of detection (LODs, S/N=3) of 0.5 μg L -1 for each and a throughput rate of 30 samples h -1 . The ITD separation was also effective for matrix isolation and reduction of interferences. Potential cationic interferences were not transferred into the anionic Cr(VI) acceptor stream. Much of the organic compounds in soil extracts were also eliminated as evidenced from standard addition and recovery studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1'-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r 2  = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans., (Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Objective: The purpose of this study is to disclose the characteristic symptoms and signs in L2, L3 and L4 nerve root disturbance., Materials and Methods: Fifty eight patients who underwent lateral herniotomy were analyzed. Breakdowns are 15 patients with L2/3 lateral disc herniation (group A), 20 patients with L3/4 lateral disc herniation (group B), and 23 patients with L4/5 lateral disc herniation (group C). The following items were examined: 1) localization of the subjective pain and numbness, 2) objective neurological findings, including deep tendon reflex, manual muscle strength (MMT), straight leg raising test (SLRT) and femoral nerve stretch test (FNST)., Results: In group A, subjective pain and/or numbness was present in the thigh aspect, proximal to the knee joint in all patients. In group B, 80% of the patients had subjective pain and/or numbness in the medial site of the knee joint. In group C, the subjective pain and/or numbness was localized in various aspects of the lower extremity. Weakness in the iliopsoas, the femoral quadriceps, and the anterior tibial muscle were observed in 60-95%, 27-70%, 0-43% of three groups, respectively. Depression or absence of the patella tendon reflex was present in 27-100% of three groups. SLRT and FNST were positive in 13-87% and 91-95% of three groups., Conclusion: Symptomatic levels of nerve root disturbance in the upper lumbar spine could not be accurately identified by objective neurological findings alone. Pain and/or numbness localized in the thigh area proximal to the knee joint is a specific sign of L2 nerve root disturbance. Either subjective pain or numbness in the medial knee joint aspect is another key sign which strongly suggests L3 nerve root disturbance., (Copyright © 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

A human small intestinal epithelial cell (HIEC) monolayer was recently established in our laboratories as a novel system to evaluate the Papp (apparent permeability coefficient) of compounds during their absorption in humans. An effusion-based analysis using polyethylene glycol oligomers with molecular weights ranging from 194-898 indicated that HIEC and Caco-2 cell monolayers both had paracellular pores with 2 distinct radiuses (∼ 5 and 9-14 Å), whereas the porosity of large pores was 11-fold higher in the HIEC monolayer (44 × 10(-8)) than in the Caco-2 cells (4 × 10(-8)). A comparison between the fraction-absorbed (Fa) values observed in humans and those predicted from Papp values in both monolayers indicated that the HIEC monolayer had markedly higher precision to predict Fa values with root mean square error of 9.40 than the Caco-2 cells (root mean square error = 16.90) for 10 paracellularly absorbed compounds. Furthermore, the accuracy of the HIEC monolayer to classify the absorption of 23 test drugs with diverse absorption properties, including different pathways in the presence or absence of susceptibility to efflux transporters, was higher than that of the Caco-2 cell monolayer. In conclusion, the HIEC monolayer exhibited advantages over Caco-2 cells in the ranking and prediction of absorption of compounds in humans., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of anticancer trifluridine (FTD; which is derived from pyrimidine nucleoside) and FTD-metabolizing enzyme inhibitor tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5. To evaluate the intestinal absorption mechanism of FTD, the uptake and transcellular transport of FTD by human small intestinal epithelial cell (HIEC) monolayer as a model of human intestinal epithelial cells was investigated. The uptake and membrane permeability of FTD by HIEC monolayers were saturable, Na(+) -dependent, and inhibited by nucleosides. These transport characteristics are mostly comparable with those of concentrative nucleoside transporters (CNTs). Moreover, the uptake of FTD by CNT1-expressing Xenopus oocytes was the highest among human CNT transporters. The obtained Km and Vmax values of FTD by CNT1 were 69.0 μM and 516 pmol/oocyte/30 min, respectively. The transcellular transport of FTD by Caco-2 cells, where CNT1 is heterologously expressed, from apical to basolateral side was greater than that by Mock cells. In conclusion, these results demonstrated that FTD exhibits high oral absorption by the contribution of human CNT1., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the in vitro and in vivo antibacterial activity of DS-8587 against multidrug-resistant (MDR) Acinetobacter baumannii. The MIC range of DS-8587 against MDR A. baumannii was 0.25-2 mg/L. These DS-8587 MICs were a minimum of 16-fold or 8-fold more potent than ciprofloxacin or levofloxacin, respectively. Bactericidal activity, a 3 log10 reduction from the initial bacterial counts, was observed within 2 h for 1593644 and 4 h for 1593684 after exposure to DS-8587. Therapeutic efficacy of DS-8587 in the murine calf muscle model was observed at 256 mg/kg. The analysis of the pharmacokinetic and pharmacodynamic index revealed that the AUC/MIC ratio showed the best correlation with efficacy. The total and free drug AUC/MIC value required for a static effect was 29.4 and 14.1, respectively. These data indicate DS-8587 would be an effective agent against MDR A. baumannii infection., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

DS-8587 is a novel broad-spectrum fluoroquinolone with extended antimicrobial activity against both Gram-positive and Gram-negative pathogens. In this study, we evaluated the antibacterial activity and mechanism of DS-8587 in 31 quinolone-resistant Acinetobacter baumannii clinical isolates. Efflux pump and qnr genes, mutations in quinolone resistance-determining regions of target enzymes, and sequence types determined by multilocus sequence typing were analyzed. Forty-two quinolone-susceptible clinical isolates were analyzed for comparison. For susceptibility testing, DS-8587 exhibited more effective antibacterial activity when compared with ciprofloxacin and levofloxacin. When combined with the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, the MIC of DS-8587 was less affected when compared with the MIC exhibited by combined ciprofloxacin and 1-(1-napthylmethyl)-piperazine. The efflux pump genes adeA/adeB/adeC and regulatory elements adeR/adeS were detected in 23 of 31 quinolone-resistant isolates. The qnrA/qnrB/qnrS genes were not detected in any A. baumannii isolates analyzed. Mutations in quinolone resistance-determining regions were observed in all 31 quinolone-resistant isolates. Multilocus sequence typing analyses revealed that 22 of 31 quinolone-resistant isolates belonged to ST-2, corresponding to international clonal lineage II. In conclusion, DS-8587 exhibits potent antibacterial activity against quinolone-resistant A. baumannii isolates that harbor mutations in quinolone resistance-determining regions. In the presence of the efflux pump inhibitor 1-(1-napthylmethyl)-piperazine, no significant changes were observed in the MIC for DS-8587. DS-8587 should be considered as a treatment option for A. baumannii including ST-2 strains that are predominant among the quinolone-resistant A. baumannii isolates found in Japan., (Copyright © 2013 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

An in vitro-in vivo correlation analysis between observed and predicted metabolic clearance in multiple preclinical species including dogs and monkeys constitutes an integral part of prediction for the pharmacokinetics in humans by using liver-derived in vitro preparations. Empirical values of the scaling factor for the extrapolation of metabolic (intrinsic) clearance in the in vitro preparation to that for whole liver were calculated for each preparation of 8 and 5 cryopreserved dog and monkey hepatocytes, respectively, by optimizing the objective function of average fold error between predicted and observed metabolic (intrinsic) clearance for eight and 11 standard compounds for dogs and monkeys, respectively. Thus obtained values of the scaling factor ranged from 5.46 × 10(9) to 19.9 × 10(9) cells/kg body weight with an average of 10.3 × 10(9) cells/kg body weight in dogs, and the value ranged from 2.36 × 10(9) to 4.21 × 10(9) cells/kg body weight with an average of 3.17 × 10(9) cells/kg body weight in monkeys, which were both consistent with biologically calculated values in corresponding species. These results demonstrated the utility of commercially available cryopreserved preparations of dog and monkey hepatocytes for the in vitro-in vivo correlation analyses with the aid of empirically or biologically obtained scaling factors at the early development stage of new drug candidates.

Pure trigeminal motor neuropathy is a very unusual disease that is characterized by trigeminal motor paralysis without trigeminal sensory disturbances and without the involvement of the other cranial nerves. We report a case of pure trigeminal motor neuropathy in a 70-year-old woman. The diagnosis was based on the results of clinical, electromyographic, and radiologic examinations. Only the motor branch of the left trigeminal nerve was damaged. Atrophy of the left-side masticatory muscles and jaw resulted in facial asymmetry. Magnetic resonance imaging (MRI) of the head and face did not detect any pathologic lesion, with the exception of atrophy and fatty infiltration of the muscles innervated by the left trigeminal motor nerve. The etiology of the patient's pure trigeminal motor neuropathy was undetermined. Patients with suspected trigeminal motor neuropathy should undergo MRI of the head and face to evaluate the sequelae of denervation and to detect an intracranial or extracranial lesion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: The aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix., Main Methods: After obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24-48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting., Key Findings: LPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each., Significance: This study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A method for the stereoselective determination of D- and L-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H(3,)(82)Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for D- and L-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for D-selenomethionine and 6% and 3% for L-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for D-selenomethionine and 2% and 1% for L-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aims: Magnesium (Mg) deficiency has been reported to be associated with the development of the metabolic syndrome, cardiovascular diseases, and sudden death. We examined the influence of chronic Mg deficiency on cardiac tolerance to hypoxia/reoxygenation injury., Main Methods: Mice were fed an Mg-deficient diet for 4 weeks, and then their hearts were excised for Langendorff perfusion experiments. The levels of total Mg in the blood and heart were quantified by atomic absorption spectrometry., Key Findings: In Mg-deficient mice, the Mg concentration in whole blood was markedly decreased; however, that in the heart remained unchanged. When the hearts of control mice were exposed to hypoxia/reoxygenation, removal of extracellular Mg from a normal Krebs solution containing 1.2 mM Mg resulted in a significant decrease in the recovery of the tension-rate product (TRP) upon reoxygenation. In Mg-deficient mice, the recovery of TRP in the heart was reduced significantly in the absence of extracellular Mg compared to that in controls. The addition of Mg to the perfusate did not improve TRP recovery. During hypoxia/reoxygenation, cardiac damage evaluated by myocardial aspartate amino transferase (AST) release was greater in hearts of Mg-deficient mice than in that of control mice., Significance: These results indicate that chronic Mg deficiency causes severe hypomagnesemia and a decrease in cardiac tolerance to hypoxia, without changing the intracellular Mg content. The decreased tolerance to hypoxia was not affected by the presence or absence of extracellular Mg, suggesting that some intracellular metabolic abnormalities develop in the cardiac myocytes of Mg-deficient mice., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Introduction: Understanding the mechanisms of drug resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is essential to develop novel chemotherapies for non-small cell lung cancer (NSCLC). Therefore, we analyzed the expression and function of ATP-binding cassette (ABC) transporters in EGFR TKI-resistant NSCLC., Methods: In three newly established AG1478-resistant NSCLC cell lines, we evaluated the expression profile of ABC transporters and genotyping of ABCG2 by real-time polymerase chain reaction and elucidated their function by Hoechst dye efflux analyses. The growth-inhibitory effect of the topoisomerase I inhibitor Hoechst 33342, which is extruded by ABCG2, was also investigated in these cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay., Results: In AG1478-resistant cells, significantly less ABCG2 was expressed, and the ratios of the cells with a strong ability to extrude Hoechst dye were remarkably smaller than in the parent cells. Because of the ABCG2 downregulation and loss of function due to C421A/C421A homozygosity, PC-14AG50R was thus considered to be more sensitive to Hoechst 33342 than the parental cells. All AG1478-resistant cells were more sensitive to the combination of Hoechst 33342 and AG1478 than to single agent., Conclusions: Resistance to EGFR TKI in NSCLC is associated with the downregulation of ABCG2 expression. A topoisomerase I inhibitor alone or in combination with EGFR TKI might offer a promising strategy for treating NSCLC that is resistant to EGFR TKI.

The calcitonin gene-related peptides (CGRP), alphaCGRP and betaCGRP, have been implicated to play various roles in primates and rodent. However, since the expression information has been limited, in the present study, we measured the amount of gene expression in mouse brain, liver, kidney, heart, and testis at embryonic day (E) 14, E17, postnatal day (P) 1, P7, and adult using real-time PCR, and determined the precise localization of alphaCGRP and betaCGRP sense/antisense transcripts in tissues using in situ hybridization. The sense transcripts of alphaCGRP and betaCGRP were found mainly in brain, and their amount profiles were similar in the course of development: one expression peak was observed at E17 and the other at P7. The amounts of alphaCGRP transcripts were greater than those of betaCGRP transcripts in the range between 3.6 and 31 times. In the E17 and P7 brains, the localization pattern of alphaCGRP sense transcripts was similar with that of alphaCGRP antisense transcripts. Fewer transcripts were found in neuroblasts of E17 corpus callosum, and neuroblasts of P7 corpus callosum, olfactory bulb, plexus chorioideus, and ventriculus lateralis than in other brain areas. The localization pattern of betaCGRP sense and antisense transcripts was similar to that for alphaCGRP except that the betaCGRP antisense transcripts showed spot-like localizations. Additionally, the alphaCGRP sense transcript, and betaCGRP sense and antisense transcripts were found in parafollicular cells (C cells) of E17 thyroid lobe. These findings together indicate that alphaCGRP and betaCGRP have their own roles in the ontogenic process.

Trace amounts of L-cysteine can function as a trigger, i.e., reaction initiator, in the autocatalytic sodium sulfite/hydrogen peroxide reaction system. Rapidly changing of pH after induction time is visually confirmed by color changing of bromothymol blue in this autocatalytic reaction. Based on this finding, microg L(-1) levels of L-cysteine were measured over time using the autocatalytic reaction system. The determination range using the above method was 5.0 x 10(-8)-2.5 x 10(-6)M, the detection limit (3 sigma) was 1.8 x 10(-8)M (1.94 microg L(-1)), and the relative standard deviation was 2.41% at an l-cysteine concentration of 5 x 10(-7)M (n=5). This method was also applied to length detection-flow injection analysis. The determination range for the flow injection analysis was 2.0 x 10(-7)-1.0 x 10(-5)M. The detection limit (3 sigma) was 1.4 x 10(-7)M (17.0 microg L(-1)), and the relative standard deviation was 0.91% at an initial L-cysteine concentration of 10(-6)M (n=5).

A trapping approach for semi-quantitative assessment of bioactivation potential has been established for new chemical entities by using [(35)S]cysteine and [(14)C]sodium cyanide as trapping reagents. Reactive metabolites were trapped as radioactive adducts with the trapping reagents to be analyzed by radio-LC(/MS). As a reference, hepatotoxic drugs (clozapine, diclofenac, R-(+)-pulegone and troglitazone) were tested in the [(35)S]cysteine trapping assay and the proposed structures of the cysteine adducts were consistent with glutathione adducts previously reported. The accuracy of this methodology to predict bioactivation potential of structurally diverse non-radiolabeled test compounds was evaluated by comparing the radiochromatographic peak area obtained in this assays with the extent of covalent binding to protein assessed by the conventional method using radiolabeled test compounds. The value obtained from the [(35)S]cysteine trapping assay in human liver microsomes predicted potential for covalent binding of the test compounds to proteins with reasonable accuracy. A combination of trapping reagents ([(35)S]cysteine and [(14)C]cyanide) improved the accuracy for prediction of bioactivation potential by simultaneously trapping both types of electrophilic reactive metabolites. This method is expected to be a useful to prioritize compounds for further development based on the bioactivation liability, especially at the lead optimization stage.

Background/aims: Mortality after 90% partial hepatectomy in mice was associated with severe acute liver failure. Recently, we revealed that platelets have a strong promotional effect on hepatic regeneration. In the present study, we investigated the effect of thrombocytosis on liver regeneration after 90% hepatectomy in mice., Methods: For thrombocytosis induction PEG-rHuMGDF was injected 5 days before operation. Hepatectomy, sparing only the caudate lobe, was performed in normal and thrombocytotic BALB/c mice. Survival rate, platelet number, liver weight/body weight ratio, proliferating cell nuclear antigen, serum parameters, signal transduction and overexpressed genes were examined., Results: Platelet number was significantly higher in thrombocytotic group. All mice in normal group died within 30 h after hepatectomy. Survival rate in thrombocytotic group was 6/11 at 30 h and 3/11 one week after hepatectomy. Activation of Akt and STAT3 signaling pathways in thrombocytotic group was observed earlier and recognized to be stronger compared to normal group. Cell cycle, signaling pathways, metabolism and transport genes were significantly overexpressed in thrombocytotic group up to 24h after hepatectomy., Conclusions: Under the thrombocytotic condition, liver regeneration occurred even in 90% hepatectomized mice. Platelets contribute to cell cycle progression and metabolic pathways in addition to preventing acute liver failure.

Introduction: Accelerating the speed of orthodontic tooth movement should contribute to the shortening of the treatment period. This would be beneficial because long treatment times are a negative aspect of orthodontic treatment. In this study, we evaluated the effects of mechanical stimulation by resonance vibration on tooth movement, and we showed the cellular and molecular mechanisms of periodontal ligament responses., Methods: The maxillary first molars of 6-week-old male Wistar rats were moved to the buccal side by using an expansive spring for 21 days (n = 6, control group), and the amount of tooth movement was measured. Additional vibrational stimulation (60 Hz, 1.0 m/s(2)) was applied to the first molars by using a loading vibration system for 8 minutes on days 0, 7, and 14 during orthodontic tooth movement (n = 6, experimental group). The animals were killed under anesthesia, and each maxilla was dissected. The specimens were fixed, decalcified, and embedded in paraffin. Sections were used for immunohistochemical analysis of receptor activator of NF kappa B ligand (RANKL) expression. The number of osteoclasts in the alveolar bone was counted by using TRAP staining, and the amount of root resorption was measured in sections stained with hematoxylin and eosin., Results: The average resonance frequency of the maxillary first molar was 61.02 +/- 8.38 Hz. Tooth movement in the experimental group was significantly greater than in the control group (P <.05). Enhanced RANKL expression was observed at fibroblasts and osteoclasts in the periodontal ligament of the experimental group on day 3. The number of osteoclasts in the experimental group was significantly increased over the control group on day 8 (P <.05). Histologically, there were no pathological findings in either group or significant differences in the amount of root resorption between the 2 groups., Conclusions: The application of resonance vibration might accelerate orthodontic tooth movement via enhanced RANKL expression in the periodontal ligament without additional damage to periodontal tissues such as root resorption.

A novel method for the prediction of drug-drug interaction has been established based on the in vitro metabolic stability in the "serum incubation method" using cryopreserved human hepatocytes suspended in 100% human serum. As a novel approach, the inhibitory effect of inhibitors on the metabolism of substrates during the first-pass elimination process in the liver (hepatic availability) and in the elimination process from the systemic circulation (hepatic clearance) were separately predicted with the anticipated inhibitor/substrate concentrations during absorption and in the systemic circulation, respectively. Ketoconazole strongly inhibited CYP3A4-mediated terfenadine metabolism in vitro, and the method predicted 6- to 37-fold increase of terfenadine AUC by the concomitant dosing of ketoconazole, which reasonably well agreed with the observed 13- to 59-fold increase of AUC in clinical studies. The CYP3A4-mediated metabolism of indinavir was also subject to the inhibition by ketoconazole in vitro at the lower indinavir concentration (2 microM), whereas no substantial inhibition was observed at 12 microM due to the saturation of indinavir metabolism. Predicted no interaction between ketoconazole and indinavir was consistent with the minimal increase (1.3-fold increase) of indinavir AUC by ketoconazole observed in clinical study. In addition, the method was applied to the CYP2D6-mediated desipramine-quinidine interaction: the predicted 6.4-fold increase of desipramine AUC by quinidine was consistent with the observed 6.7-fold increase of AUC in the clinical drug-drug interaction study. On the other hand, desipramine metabolism was little affected by ketoconazole in vitro, and consequently, it predicted no drug-drug interaction between desipramine and ketoconazole in humans, which agreed with the negligible interaction observed in clinical study. The accuracy of predictions for drug-drug interaction by the serum incubation method was evaluated by comparing the predicted increase of AUC after an oral administration by the inhibitor with the corresponding drug-drug interaction reported from clinical studies. These data demonstrated that the newly established method provides an in vitro tool for the prediction of drug-drug interaction with the accuracy ranging from 0.46 to 1.5.

Wilson's disease is an inherited, autosomal recessive disorder of copper accumulation and toxicity. Lifelong chelation therapy is essential in all Wilson's disease patients. Intestinal absorption of some compounds is limited partly because they are preferentially transported in the secretory direction. Several ATP-binding cassette (ABC) transporters are expressed in the apical membrane of the small intestine and secrete various drugs into the lumen. In this study, we investigated the characteristics of the intestinal efflux ABC transporters in LEC rats. We found that the expression of multidrug resistance-associated protein 2 (Mrp2) in the jejunum of Long-Evans Cinnamon (LEC) rats, an animal model for Wilson's disease, is decreased.

The liver plays important roles in the detoxification of xenobiotics. Hepatobiliary transporters contribute to hepatic uptake and efflux processes of xenobiotecs. Expressions of these transporters may be modulated under the condition of hepatic failure. Long-Evans Cinnamon (LEC) rats provide a pertinent model for basic and clinical studies on hepatitis. However, only a few reports describing the properties of hepatobiliary transporters in LEC rats have appeared in the literature. We investigated the expression levels of hepatobiliary transporters in LEC rats by real-time RT-PCR. We found that hepatic expressions of three sinusoidal organic anion transporters, Ntcp, Oatp1a1 and Oatp1a4, were decreased in LEC rats. However, no significant difference of the expressions of Mrp2 and Bsep, organic anion transporters located on canalicular membrane, were found between Wistar rats and LEC rats.

Hospital-acquired legionellosis is one of the serious problems in nosocomial infection. For risk assessment of nosocomial Legionella infection, we surveyed samples from bathrooms for public use in three hospitals and two nursing homes to determine whether Legionella pneumophila was present. A total of 70 hot bathwater samples and samples wiped from bathtubs were collected at 1-h intervals. Fifteen shower-water and 15 inner-head samples were obtained at the start of a bath. Water samples were cultured using the Legionella spp. selective medium, and discrimination between L. pneumophila and other Legionella spp. was performed by PCR analysis. L. pneumophila serogroup 7 was detected in 1 bathwater and 1 wiped sample, both of which were collected 1 h after daily use from the same bathtub in a hospital. However, L. pneumophila SG7 was not detected in any other samples. Furthermore, the concentrations of free residual chlorine in most bath- and shower-water samples were lower than 0.1 mg/l. These results suggest that L. pneumophila has become a potential pathogen for nosocomial infections in public-type hospital baths. From the point of view of an infection-control program, it might be advisable to hold the concentration of free residual chlorine at 0.2-0.4 mg/l, which is generally required for public baths in Japan.

It is known that secretory transport limits the oral bioavailability of certain drugs. However, there is little information on the secretion of anionic compounds in the intestine. Phenolsulfonphthalein (PSP) and p-aminohippuric acid (PAH) have been used widely as substrates for organic anion transport systems. PAH is transported in the secretory direction in the intestine. It is possible that PSP and PAH share the same transport system at the mucosal membrane. The purpose of this study was to characterize the transport system for PSP in the intestine. In the jejunum, the serosal-to-mucosal permeation rate of PSP was significantly reduced in an ATP-depleted condition, whereas a significant difference was not observed in the ileum. Some multidrug resistance-associated protein 2 (Mrp2) inhibitors inhibited PSP permeation in the jejunum. However, pravastatin, a substrate of Mrp2, did not inhibit the PSP permeation. The jejunal secretory transport of pravastatin was significantly reduced in an ATP-depleted condition and by addition of probenecid, but PSP did not affect the jejunal permeation of pravastatin. These results suggest that PSP is secreted into the intestinal lumen by Mrp2-like transporter and that two Mrp2 substrates, PSP and pravastatin, are likely to be transported by different transport systems at the mucosal membrane.

This study describes a new simultaneous determination of haloperidol and bromperidol and their reduced metabolites by modification of automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1ml of plasma using chloroform-hexane (30:70 (v/v)), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C(18) analytical column for separation. The mobile phases consisted of phosphate buffer (0.02M, pH 4.6), perchloric acid (60%) and acetonitrile (54:1:45 (v/v)) and was delivered at a flow-rate of 0.6ml/min. The peak was detected using a UV detector set at 215nm. The method was validated for the concentration range 1-100ng/ml, and good linearity (r >0.999) was confirmed. Intra-day coefficient variations (CVs) for haloperidol, reduced haloperidol, bromperidol and reduced bromperidol were less than 2.5, 3.1, 2.4 and 2.5%, respectively. Inter-day CVs for corresponding compounds were 3.9, 5.1, 2.6 and 4.4%, respectively. Relative errors ranged from -5 to 10% and mean recoveries were 96-100%. The limit of quantification was 1.0ng/m for each compound. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring, particularly in patients receiving both haloperidol and bromperidol.

We examined the mechanisms responsible for the production of fibronectin induced by oxidized low-density lipoprotein (oxLDL) in rat mesangial cells. oxLDL accelerated the production of fibronectin with the preceding generation of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine suppressed the oxLDL-induced fibronectin production as well as ROS generation. oxLDL also elicited the activation of SP-1, nuclear factor-kappaB, and cAMP response element-binding protein, but not activator protein-1. Among these activated transcription factors, N-acetylcysteine inhibited the activation of SP-1 only. 7-Ketocholesterol, an oxidized lipid in oxLDL particles, induced the production of fibronectin and the activation of SP-1, those which were suppressed by N-acetylcysteine. Furthermore, mithramycin A, an inhibitor of SP-1, also suppressed the oxLDL- and 7-ketocholesterol-stimulated production of fibronectin. These results suggest that oxLDL stimulates fibronectin production, at least in part, through the ROS-dependent activation of SP-1 in rat mesangial cells, and further that the ROS-dependent cellular responses may be elicited by 7-ketocholesterol.

A healthy 6-year-old boy had complained of fever and chest pain for 3 days. On admission, he had a mass on the sternum, 3.7 x 2.5 cm in size. Abnormal laboratory findings included a white blood cell count of 12,900/microl, erythrocyte sedimentation rate (ESR), 74 mm/h, and C-reactive protein (CRP), 9.7 mg/dl. Ultrasound examination of the chest revealed a hypoechoic lesion on the sternum that was 30 x 15 mm in size. Chest computed tomography (CT) scan showed no bone fracture or bone erosion. The patient received cefpirome, given intravenously at 60 mg/kg per day for 10 days. Incision and drainage was performed on the seventh day in the hospital, and we collected 0.5 ml of pus. Salmonella enteritidis was detected from the drainage. However, the patient had no gastrointestinal symptoms. He was discharged on the fourteenth hospital day, as he was asymptomatic. Results of all physical and laboratory examinations including blood and stool cultures and ultrasound examinations, were within the normal limits upon discharge.

Treatment of 3-C-beta-D-galactopyranosylphloroacetophenone in hot water with a catalytic amount of p-toluenesulfonic acid afforded a spiroketal compound as the main product. The chirality of the spiro carbon of the product was R, which is the opposite of the spiroketal obtained by the conversion of 3-C-beta-D-glucopyranosyl phloroacetophenone under identical conditions. The structure was determined by 1H-1H COSY, 1H-13C COSY, NOESY and HMBC spectroscopy.

Investigations of platelet adhesion to adhesive proteins have been pursued to understand the basic mechanisms of hemostasis and thrombosis. Most assays used to determine platelet adhesion under stasis conditions rely on radiolabeled platelets. We describe a new microtiter immunoassay to study platelet adhesion to adhesive proteins under stasis conditions. Direct comparison of platelet adhesion to fibronectin using a standard platelet adhesion assay based on 51Cr-labeled platelets and the new immunoassay showed that the optical density values obtained with the immunoassay are directly proportional to the number of platelets bound. The choice of platelet suspension buffer crucial for the design of such experiments, because the adhesion of resting platelets to fibronectin is increased in response to thrombin stimulation. This increase buffer rather than Tris buffer. Platelet adhesion to fibronectin is increased in response to thrombin stimulation. This increase can be inhibited by synthetic RGD peptides. The thrombin-induced increase of platelet adhesion to fibronectin could be detected with antibodies against actin and glycoprotein IIb-IIIa, but not against the alpha-granule constituent platelet factor 4 (PF4). This assay is very versatile, because it avoids the use of radioactivity, and allows the parallel processing of a large number of samples. In addition, the parallel use of antibodies against different platelet antigens allows the screening for platelet activation events associated with the measured platelet adhesion.

During muscle contraction, chemical energy from ATP hydrolysis is converted to the relative sliding movement of actin and myosin filaments. In order to elucidate the molecular mechanism of sliding-force generation, it is a crucial clue to know an elastic modulus of myosin. Here, we report direct measurements of Young's modulus of myosin head (myosin subfragment-1) isolated from rabbit skeletal muscle, using a surface forces apparatus. Our results show that the elasticity of myosin subfragment-1 has direction and is about 0.3 GPa along the long axis during ATP hydrolysis. When the bow-shaped subfragment-1 is modelled as an elastic rod, the stiffness and the bending fluctuations of subfragment-1 are calculated to be 3-7 pN/nm and about 1 nm, respectively. These results strongly support a model of multiple power strokes rather than the conventional tilting-crossbridge model.

This study examined the peripheral endothelium-dependent vasodilatory response to acetylcholine and the endothelium-independent vasodilatory response to nitroprusside in 19 patients with chronic heart failure and eight controls. These peripheral blood flow responses were compared with hyperemic calf blood flow changes after maximum leg exercise and 5-min femoral occlusion. The peripheral blood flow response to forearm intra-arterial infusion of acetylcholine and sodium nitroprusside, and reactive hyperemic calf blood flow changes were measured by plethysmography. All peripheral blood flow responses were significantly reduced in patients with chronic heart failure (P < 0.05). Reduction of acetylcholine-mediated changes in peripheral blood flow was correlated with exercise-induced calf blood flow response (r = 0.51, P < 0.05), but not with occlusion-induced calf blood flow response (r = 0.02, NS). Sodium nitroprusside-mediated changes were not correlated with any reactive hyperemic blood flow responses (exercise: r = 0.27, NS; occlusion: r = 0.11, NS). When the patients were divided into two subgroups based on the median exercise-induced calf blood flow change, the subgroup with the lower calf blood flow response showed a reduction in exercise capacity (anaerobic threshold: 11.8 +/- 0.6 vs. 14.6 +/- 1.0 ml/kg/min; P < 0.05). These findings suggest that endothelial dysfunction is related to a decrease in exercise-induced skeletal muscle blood flow and exercise capacity in patients with chronic heart failure.

Sulfate conjugates are a heterogeneous class of polar, anionic metabolites that result from the conjugation of endogenous and exogenous compounds. Sulfate conjugates exhibit a high degree of binding to albumin, the extent of which usually exceeds those of their parent compounds. Preponderant direct and indirect evidence suggests that sulfation activity is slightly higher in the periportal than in the perivenous (centrilobular) region of the liver, but recent immunohistochemical studies imply that specific isoforms of the sulfotransferases may also be preferentially localized in the perivenous region. Entry of sulfate conjugates into the liver cell is poor unless discrete carriers are present. Although known transport carriers exist for the sulfated bile acids, the specificity of the carriers for drug sulfate conjugates is presently unknown. The removal of sulfates is usually by way of biliary excretion while, on occasion, sulfates can be desulfated and participate in futile cycling with their parent compounds. The binding, transport, and hepatic elimination of various drug sulfate conjugates are examined. Non-recirculating studies carried out in the perfused rat liver with the multiple indicator dilution technique under varying input sulfate conjugate concentrations have provided essential information on the effects of vascular (red blood cells and plasma protein) binding on transport and removal of the conjugates. These studies clearly demonstrate the need to study protein binding, transmembrane transfer characteristics across the liver basolateral (sinusoidal) and canalicular membranes, and enzyme zonation in a distributed-in-space fashion in order to properly define the handling of sulfate conjugates in the liver.