MpFAE3, a beta-ketoacyl-CoA synthase gene in the liverwort Marchantia polymorpha L., is preferentially involved in elongation of palmitic acid to stearic acid

Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50% of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.     

Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.     

A Bioluminescence Assay Using Nitrosomonas europaea for Rapid and Sensitive Detection of Nitrification Inhibitors

An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O₂ uptake and NO₂⁻ production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

Extracting and understanding the relationship between components of the capacity in elderly people in terms of electrical device comprehension

The purpose of this study is to extract components of the capacity which elderly people have in comprehending electrical devices and determine its relationship with the components. Initially, we proposed a hypothesis through examining previous studies. The hypothesis states that the capacity which elderly people have mainly consists of four components, i.e., motivation, working memory, logical thinking and experience with personal computers (PC) or mobile phones. Then, some tests were conducted to examine the hypothesis. In this research, elderly people were interviewed about their impressions and experience with electrical devices. Moreover, three tests were conducted including, card sorting, tasks using digital video cameras and a test to measure working memory. As analysis methods, the Quantification 1 was used to see which component was important. In addition, Boolean algebra was conducted to simplify the components and understand some relationships. As a result, a relationship with the components in electrical devices was revealed. Furthermore, the use of Boolean algebra and the Quantification 1 suggested that the experience with PCs or mobile phones was the most important component for elderly people.     

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.     

Enteric absorption and pharmacokinetics of oseltamivir in critically ill patients with pandemic (H1N1) influenza

Background

Whether the enteric absorption of the neuraminidase inhibitor oseltamivir is impaired in critically ill patients is unknown. We documented the pharmacokinetic profile of oseltamivir in patients admitted to intensive care units (ICUs) with suspected or confirmed pandemic (H1N1) influenza.

Methods

We included 41 patients 18 years of age and older with suspected or confirmed pandemic (H1N1) influenza who were admitted for ventilatory support to nine ICUs in three cities in Canada and Spain. Using tandem mass spectrometry, we assessed plasma levels of oseltamivir free base and its active metabolite carboxylate at baseline (before gastric administration of the drug) and at 2, 4, 6, 9 and 12 hours after the fourth or later dose.

Results

Among the 36 patients who did not require dialysis, the median concentration of oseltamivir free base was 10.4 (interquartile range [IQR] 4.8–14.9) μg/L; the median concentration of the carboxylate metabolite was 404 (IQR 257–900) μg/L. The volume of distribution of the carboxylate metabolite did not increase with increasing body weight (R² = 0.00, p = 0.87). The rate of elimination of oseltamivir carboxylate was modestly correlated with estimations of creatinine clearance (R² = 0.27, p < 0.001). Drug clearance in the five patients who required continuous renal replacement therapy was about one-sixth that in the 36 patients with relatively normal renal function.

Interpretation

Oseltamivir was well absorbed enterically in critically ill patients admitted to the ICU with suspected or confirmed pandemic (H1N1) influenza. The dosage of 75 mg twice daily achieved plasma levels that were comparable to those in ambulatory patients and were far in excess of concentrations required to maximally inhibit neuraminidase activity of the virus. Adjustment of the dosage in patients with renal dysfunction requiring continuous renal replacement therapy is appropriate; adjustment for obesity does not appear to be necessary.A substantial number of cases of pandemic (H1N1) influenza have involved young adults and adolescents without serious comorbidities who present with severe viral pneumonia complicated by acute respiratory distress syndrome, rhabdomyolysis, renal failure and, occasionally, shock.^1,2 Antiviral therapy in such critically ill patients typically requires oral or nasogastric administration of the neuraminidase inhibitor oseltamivir. Current guidelines from the World Health Organization for the pharmacologic management of progressive or severe pandemic (H1N1) influenza recommend the consideration of high-dose therapy (≥ 150 mg twice daily).^3,4 Critically ill patients exhibit defects in gastrointestinal absorption because of impaired gut perfusion, edema of the bowel wall and ileus as a consequence of critical illness and shock.⁵ Whether the enteric absorption of oseltamivir is impaired in such patients is unknown.We undertook this study to document the pharmacokinetic profile of oseltamivir administered orally or by nasogastric tube in patients admitted to intensive care units (ICUs) with respiratory failure due to suspected or confirmed pandemic (H1N1) influenza.     

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.     

DNA repair defect in AT cells and their hypersensitivity to low-dose-rate radiation

Ataxia telangiectasia (AT) and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative conditions with high- (2 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. While normal cells showed a higher resistance after irradiation at a low dose rate than a high dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. Although the frequency of micronuclei induced by low-dose-rate radiation was greatly reduced in normal cells, it was not reduced significantly in AT cells. The number of gamma-H2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few gamma-H2AX foci were observed after low-dose-rate irradiation in normal cells, significant and dose-dependent numbers of gamma-H2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. Significant phosphorylation of ATM proteins was detected in normal cells after low-dose-rate irradiation, suggesting that the activation of ATM plays an important role in the repair of DNA damage during low-dose-rate irradiation. In conclusion, AT cells may not be able to repair some fraction of DNA damage and are severely affected by low-dose-rate radiation.