Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.     

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.     

Identification of eight members of the Argonaute family in the human genome

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.     

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays

Culture of human cells with human interferon alpha and beta (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF(r) and F-IF(r) cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.     

Search for UV-responsive genes in human cells by differential mRNA display: involvement of human ras-related GTP-binding protein, Rheb, in UV susceptibility

We have previously demonstrated that glucose deprivation alters the glycosylation of the GLUT1 glucose transporter in 3T3-L1 adipocytes. Many aberrantly glycosylated proteins are retained in the endoplasmic reticulum by interaction with chaperones. Herein, we use three independent procedures to show that GLUT1 is targeted to the plasma membrane, despite alterations in glycosylation. While earlier experiments revealed that plasma membrane targeting of aglyco GLUT 1 transporter was significantly reduced, our data show for the first time that altered glycosylation provides sufficient information to drive appropriate trafficking.     

Influence of temperature on activity of the isolated whole bladder preparation of neonatal and adult rats

The temperature sensitivity of in vitro whole bladder preparations from neonatal and adult rats with or without chronic partial urethral obstruction was investigated. After the bladder was filled to a volume eliciting isovolumetric contractions, temperature was changed between 19 and 38 degrees C. In all preparations, higher temperatures were associated with higher frequencies of spontaneous intravesical pressure waves (IVPW). In 1- to 2-wk-old neonates, IVPW amplitude increased as the temperature increased; however, in older neonates and normal adults, the opposite occurred. The transition period was at 3 wk of age when bladder volume also markedly increased. At this age as well as in adult rats with outlet obstruction, changing temperature had little influence on the amplitude of IVPW. Thus obstructed outlet bladders and 3-wk-old bladders had similar properties. It is concluded that the properties of bladder muscle are changed during postnatal maturation and that in 3-wk-old rats, when brain control of voiding is emerging, micturition is abnormal, leading to obstructive changes in bladder muscle.     

Search for gravity-responsive genes by PCR-based mRNA differential display in human cells

Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].     

Probucol-Induced α-Tocopherol Deficiency Protects Mice against Malaria Infection

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.     

Association between delayed bedtime and sleep-related problems among community-dwelling 2-year-old children in Japan

Background

Although delayed sleep timing causes many socio-psycho-biological problems such as sleep loss, excessive daytime sleepiness, obesity, and impaired daytime neurocognitive performance in adults, there are insufficient data showing the clinical significance of a ‘night owl lifestyle’ in early life. This study examined the association between habitual delayed bedtime and sleep-related problems among community-dwelling 2-year-old children in Japan.

Methods

Parents/caregivers of 708 community-dwelling 2-year-old children in Nishitokyo City, Tokyo, participated in the study. The participants answered a questionnaire to evaluate their child’s sleep habits and sleep-related problems for the past 1 month.

Results

Of the 425 children for whom complete data were collected, 90 (21.2%) went to bed at 22:00 or later. Children with delayed bedtime showed significantly more irregular bedtime, delayed wake time, shorter total sleep time, and difficulty in initiating and terminating sleep. Although this relationship indicated the presence of sleep debt in children with delayed bedtime, sleep onset latency did not differ between children with earlier bedtime and those with delayed bedtime. Rather, delayed bedtime was significantly associated with bedtime resistance and problems in the morning even when adjusting for nighttime and daytime sleep time.

Conclusions

Even in 2-year-old children, delayed bedtime was associated with various sleep-related problems. The causal factors may include diminished homeostatic sleep drive due to prolonged daytime nap as well as diurnal preference (morning or night type) regulated by the biological clock.