FMN binding and photochemical properties of plant putative photoreceptors containing two LOV domains, LOV/LOV proteins.

LOV domains function as blue light-sensing modules in various photoreceptors in plants, fungi, algae, and bacteria. A LOV/LOV protein (LLP) has been found from Arabidopsis thaliana (AtLLP) as a two LOV domain-containing protein. However, its function remains unknown. We isolated cDNA clones coding for an LLP homolog from tomato (Solanum lycopersicum) and two homologs from the moss Physcomitrella patens. The tomato LLP (SlLLP) contains two LOV domains (LOV1 and LOV2 domains), as in AtLLP. Most of the amino acids required for association with chromophore are conserved in both LOV domains, except that the amino acid at the position equivalent to the cysteine essential for cysteinyl adduct formation is glycine in the LOV1 domain as in AtLLP. When expressed in Escherichia coli, SlLLP binds FMN and undergoes a self-contained photocycle upon irradiation of blue light. Analyses using mutant SlLLPs revealed that SlLLP binds FMN in both LOV domains, although the LOV1 domain does not show spectral changes on irradiation. However, when Gly(66) in the LOV1 domain, which is located at the position equivalent to the essential cysteine of LOV domains, is replaced by cysteine, the mutated LOV1 domain shows light-induced spectral changes. In addition, all four LOV domains of P. patens LLPs (PpLLP1 and PpLLP2) show the typical features of LOV domains, including the reactive cysteine in each. This study shows that plants have a new LOV domain-containing protein family with the typical biochemical and photochemical properties of other LOV domain-containing proteins such as the phototropins.

Optimal administration of tacrolimus in reduced-size liver.

The optimal administration of immunosuppressants such as tacrolimus (Tac) for small-for-size (SFS) grafts, where the functional liver mass is small and must regenerate, has not been reported so far. The aim of this study is to clarify the characteristics of Tac metabolism according to liver volume. Seven-week-old male Wistar rats were randomly divided into three groups: (1) Tac administrated and 70% Hx group (Tac 70% Hx group), (2) Tac administrated and 90% Hx group (Tac 90% Hx group), and (3) vehicle administrated and 90% Hx group (control 90% Hx group). In both the Tac groups, Tac (0.3 mg/kg) was given daily for 3 days before operation, and daily after surgery until sacrifice (each time point; n = 5). The plasma concentration of Tac (trough level), as well as liver toxicity, were measured. The plasma concentration of Tac in the Tac 90% Hx group was significantly higher than in the Tac 70% Hx group from 24 to 72 h after operation. Furthermore, expression of CYP3AII mRNA was significantly lower in the Tac 90% Hx group than in the Tac 70% Hx group. Regarding the liver toxicity, there was no significant difference in both the Tac 90% Hx and the control 90% Hx groups. In this experimental study, the plasma concentration of Tac was dependent on the remnant liver volume. Therefore, special attention in regard to Tac administration should also be taken for patients with SFS grafts in living-donor liver transplantation (LDLT).

Relationship between decreased expression of squamous cell carcinoma antigen 2 and E-cadherin in primary cervical cancer lesions and lymph node metastasis.

Squamous cell carcinoma antigen (SCCA) has been used for the management of squamous cell carcinoma, especially in order to evaluate therapeutic effects and monitor recurrence. Recent studies have shown that SCCA performs several biological functions and can influence the behavior of cancer cells. It is well known that altered expression of E-cadherin is involved in the process of cancer invasion and metastasis. The present study was therefore undertaken to investigate the relationship between the expression of SCCA, E-cadherin and lymph node metastasis in advanced cervical squamous cell carcinoma patients. We studied 70 patients who had undergone radical hysterectomy and pelvic lymphadenectomy for stage IB, IIA and IIB of the disease, without pretreatments. Immunohistochemistry, using monoclonal antibodies against SCCA2 and E-cadherin, was performed to examine the relationship between SCCA2 and E-cadherin expression patterns in primary cancer lesions and lymph node metastasis. There was a significant positive relationship between the two expression patterns in primary cancer lesions (p<0.01). Both exhibited a heterogeneous expression pattern in the primary tumor which indicated a significant relationship with lymph node metastasis (p<0.01). Our data clearly show that SCCA2 expression is significantly related to E-cadherin expression and that the heterogeneous pattern of SCCA and E-cadherin in primary lesions is strongly associated with the high incidence of lymph node metastasis in cervical squamous cell carcinoma. These findings suggest that SCCA2 may be involved in cancer behavior such as metastasis, and as such can be a useful marker in predicting lymph node metastasis.

[The Pharmaceuticals and Medical Devices Agency's Approach to Facilitate Risk Communication and Its Challenges].

　The issue of drug lag in Japan has been rapidly reduced in recent years, and newly approved drugs now become available on Japanese and international markets at the same time. In this context, the risk management plan (RMP) system was introduced in 2012. RMPs describe important safety concerns recognized by Japan's Pharmaceuticals and Medical Devices Agency (PMDA) and marketing authorization holders (MAHs), as well as safety measures that MAHs request healthcare professionals (HCPs) to follow. The publication of RMPs is expected to support the sharing of drug risk management among HCPs during the postmarketing phase. In addition, to encourage risk communication between HCPs and patients, the PMDA website provides drug guides for patients and other information to promote proper understanding of drugs by patients and their families and enable them to identify serious adverse drug reactions at an early stage. However, the results of surveys conducted by the PMDA in FY2014 and FY2015 revealed low levels of awareness of RMPs and drug guides for patients in hospitals and other healthcare institutions. The surveys also showed that information regarding the proper use of drugs from MAHs and the PMDA was not incorporated into practice at healthcare institutions, resulting in the repeated release of identical safety alerts. To facilitate the increased utilization of risk communication tools, the PMDA has been providing and disseminating these tools through its website. This study addresses those efforts and the associated challenges.

E-cadherin increases squamous cell carcinoma antigen expression through phosphatidylinositol-3 kinase-Akt pathway in squamous cell carcinoma cell lines.

Squamous cell carcinoma antigen (SCCA) has been used for the management of squamous cell carcinoma, especially for evaluating therapeutic effects and monitoring recurrence. It has been reported that SCCA has several biological activities and influences behavior of cancer cells. E-cadherin is a cell adhesion molecule and plays important roles in the process of cancer invasion and metastasis. Our previous studies have shown that blockage of E-cadherin action by anti-E-cadherin antibody treatment suppresses SCCA production in squamous cell carcinoma cells. This finding strongly suggests that E-cadherin regulates SCCA expression. The present study was, therefore, undertaken to investigate the correlation between E-cadherin and SCCA2. For this purpose, E-cadherin cDNA was transfected into squamous cell carcinoma cell lines, SiHa and SKG IIIa. Overexpression of E-cadherin increased SCCA2 expression together with cell aggregation. We also examined the involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathway, which is one of major signaling pathways from E-cadherin. E-cadherin transfection increased phosphorylated Akt expression concomitantly with the increase in SCCA2 expression, and the increased SCCA2 expression was inhibited by a PI3K inhibitor. In conclusion, SCCA2 is up-regulated by E-cadherin through PI3K-Akt pathway, suggesting that SCCA2, as well as E-cadherin, may be involved in the regulation of cancer behavior.

Different expression patterns of intact forms of squamous cell carcinoma antigens between normal and malignant cervical squamous epithelial tissues: nondenaturing polyacrylamide gel electrophoretic analysis.

Squamous cell carcinoma antigen (SCCA), a 45-kDa tumor-associated serpin, mainly consists of two highly homologous molecules, SCCA1 and SCCA2, which possess unique proteinase inhibitory properties. Importantly, our previous study demonstrated that an intact structure of SCCAs, and not a cleaved form yielded by interacting with target proteinase, is essential for their function as a serpin. The aim of this study is therefore, to develop a simple method of analyzing expression patterns of intact forms of SCCAs (functional SCCAs) in cervical squamous epithelial tissues and to investigate whether there are any differences in the expression of intact forms of SCCAs between normal and malignant cervical squamous epithelial tissues. We used nondenaturing polyacrylamide gel electrophoresis (PAGE) with immunoblotting. The newly generated antibody, Pab Y2, recognizes only intact form of SCCAs, while the conventional antibody, Mab 27, reacts with the cleaved form of SCCA1 as well as intact forms of SCCAs. Nondenaturing PAGE using Pab Y2 showed that an intact form of SCCAs in the heat-treated tissue extract at 60 degrees C for 2 h was separated into at least five bands, termed as bands A-E from cathode to anode. By comparison with two-dimensional electrophoresis patterns of SCCAs, it was found that the first three bands, i.e. bands A-C, are derived from the intact form of SCCA1, while the other two bands, i.e. band D and E are from the intact form of SCCA2. Specifically, band E, but not band D, of SCCA2 is apparently increased in squamous cell carcinomas compared with normal squamous epithelium. In conclusion, this novel analytical approach will be useful for investigating the different expression patterns of functional SCCAs between normal and malignant cervical squamous epithelial tissues.

Carbapenem antibiotics inhibit valproic acid transport in Caco-2 cell monolayers.

The concomitant use of carbapenem antibiotics with valproic acid has been prohibited because carbapenems induced a decrease in plasma concentration of valproic acid in epileptic patients during valproic acid therapy. Our previous in vivo study in rats proposed that inhibition by carbapenem of the intestinal absorption of valproic acid might be a possible mechanism for the drug-drug interaction. To demonstrate the hypothesis, we examined the effects of imipenem and panipenem on intestinal transepithelial transport of valproic acid using Caco-2 cell monolayers. Imipenem and panipenem inhibited the transport of [14C]-valproic acid across the Caco-2 cell monolayers from apical-to-basolateral side in a concentration-dependent manner, although they had no effect on the uptake of [14C]-valproic acid by Caco-2 cells. The inhibition by the carbapenems of the valproic acid transport was found even when they were added to only the basolateral side. From these results, the carbapenems may inhibit the absorption of valproic acid at the basolateral membrane of intestinal epithelial cells, which contributes to the decrease in plasma concentration of valproic acid after oral administration.

A thick placenta: a predictor of adverse pregnancy outcomes.

PURPOSE: The aim of this study is to evaluate the efficacy of an ultrasonographic measurement of placental thickness and the correlation of a thick placenta with adverse perinatal outcome. METHODS: Placental thickness was measured in single gravidas, 16 to 40 weeks of gestation, between 2005 and 2009. Placentas were considered to be thick if their measured thickness were above the 95th percentile for gestational age. RESULTS: The incidence of thick placentas was 4.3% (138/3,183). Perinatal morbidity and neonatal conditions were worse in cases with thick placenta rather than without thick placenta. CONCLUSIONS: Ultrasonographic measurement of placental thickness is a simple method to estimate placental size. Thick placenta may be a useful predictor of adverse pregnancy outcomes.

Plasma proinflammatory and anti-inflammatory cytokine and catecholamine concentrations as predictors of neurological outcome in acute stroke patients.

PURPOSE: Proinflammatory and anti-inflammatory cytokines may play a pivotal role in cerebral inflammation, which is implicated in the development of brain injury. Systemic cytokine release is mediated by the sympathetic nervous system and catecholamines. The aim of this study was to investigate which parameters, among plasma levels of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) and the levels of the catecholamines, epinephrine and norepinephrine, contribute to the clinical outcome in acute stroke patients. METHODS: Thirty-seven acute stroke patients (ischemic, n = 19; hemorrhagic, n = 18) were enrolled. All of them were admitted to our hospital within 8 h after stroke onset. Neurological status was evaluated by a modified National Institute of Health Stroke Scale (mNIHSS) on admission and by a modified Rankin Scale (mRS) at 1 month. An mRS score of 3 or more at 1 month was considered to indicate poor outcome. Serum samples for the cytokine and catecholamine measurements were collected on admission. Plasma levels of IL-1beta, IL-6, IL-10, and TNF-alpha were determined by an enzyme-linked immunosorbent assay (ELISA) method and epinephrine and norepinephrine concentrations were determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC). RESULTS: In the ischemic stroke patients, poor outcome was noted in 9 (47%). There were no significant differences in cytokine or catecholamine concentrations between patients with poor and good outcomes, and there was no association between clinical outcome and cytokine and catecholamine concentrations. In the hemorrhagic stroke patients, poor outcome was noted in 10 (56%). IL-6 and IL-10 levels were higher in patients with poor outcome. On logistic regression analysis, higher values of IL-6 were significantly associated with clinical outcome at 1 month (odds ratio [OR], 1.25; 95% confidence interval [CI], 1.02-1.54). CONCLUSION: In ischemic stroke, plasma cytokines and catecholamines were not predictors of neurological outcome at 1 month. In hemorrhagic stroke, high levels of IL-6 in the early phase indicated a poor neurological outcome.

Calcium and reactive oxygen species mediated Zn2+ -induced apoptosis in PC12 cells.

The release of excessive Zn(2+) from presynaptic boutons into extracellular regions contributes to neuronal apoptotic events, which result in neuronal cell death. However, the mechanisms of Zn(2+)-induced neuronal cell death are still unclear. Therefore, we investigated the dynamics of intracellular Zn(2+), calcium, and reactive oxygen species in PC12 cells. The addition of Zn(2+) produced cell death in a concentration- and time-dependent manner. (45)Ca(2+) influx occurred just after the treatment with Zn(2+), although subsequent hydroxyl radical ((*)OH) production did not begin until 3 h after Zn(2+) exposure. (*)OH production was significantly attenuated in Ca(2+)-free medium or by L-type Ca(2+) channel antagonist treatment, but it was independent of the intracellular Zn(2+) content. Dantrolene treatment had no protective effects against Zn(2+)-induced cell death. Treatment with N-acetyl-L-cysteine blocked (*)OH generation and subsequent cell death. These data indicate that Ca(2+) influx and subsequent (*)OH production are critical events in Zn(2+)-induced toxicity in PC12 cells.

Hair analysis of flecainide for assessing the individual drug-taking behavior.

OBJECTIVE: The concentration of flecainide in hair was measured to determine its value as an index of individual drug-taking history. METHODS: Hair samples obtained from 15 patients treated with flecainide for more than 1 month were cut into 1-cm-long portions successively from its scalp end. The concentration of flecainide in each hair portion was measured using high-performance liquid chromatography. RESULTS: Flecainide was detected in the 1-cm-long hair portion at the scalp end in the concentration range of 38.0-411.9 ng x mg(-1), which significantly correlated with the area under the plasma flecainide concentration versus time curve. The axial centimeter-by-centimeter distribution of flecainide along the hair shaft well reflected the individual history of drug use. CONCLUSION: The present study suggests the usefulness of determining flecainide in hair to provide retrospective information on the individual drug-taking behavior qualitatively.