Association of the colistin resistance gene mcr-1 with faecal pollution in water environments in Hanoi, Vietnam.

Colistin is one of the antibiotics of last resort for human health. However, the dissemination of the plasmid-mediated colistin resistance gene mcr-1 is of great concern globally. In the One Health framework, the environment is an important component for managing antimicrobial resistance. However, little information is available concerning the prevalence of mcr-1 in water environments. We aimed to reveal the prevalence of mcr-1 in different water environments in Hanoi, Vietnam. Quantitative PCR was applied to detect mcr-1 in four urban drainages receiving untreated domestic wastewater, three rivers, five lakes and two groundwater samples. Urban drainages contained higher concentrations of mcr-1, suggesting that urban residents carry the gene. The class 1 integron-integrase gene was identified as a good surrogate of antibiotic resistance genes including mcr-1. A significant correlation was found between the levels of mcr-1 and the human-specific cross-assembly phage, which is an indicator of human faecal pollution. These results indicated that the primary source of mcr-1 in urban water environments is human faeces, which is consistent with the fact that most domestic wastewater is untreated in Hanoi. The control of untreated wastewater is critical for alleviating the spread of mcr-1 in water environments in Vietnam.

Ozone disinfection kinetics of poliovirus 1 determined by cell culture assay, RT-qPCR and ethidium monoazide qPCR reduction in a continuous quench-flow reactor.

AIMS: A continuous quench-flow (CQF) reactor was developed to collect samples at the reaction times of less than one second. The reactor is applied to determine ozone disinfection kinetics of poliovirus and to study whether EMA-qPCR can assess the viral infectivity after ozone disinfection. METHODS: Ozone disinfection of poliovirus was conducted in the developed CQF, and the disinfection kinetics were tested in the range of 0·7-5·0 s at ozone concentration of 0·08 and 0·25 mg l-1 . Inactivation, damage on viral genome and damage on capsid integrity were determined by plaque assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and ethidium monoazide treatment coupled with RT-qPCR (EMA-qPCR), respectively. RESULTS: By using CQF, 2·18 and 2·76 log10 reductions were observed at the reaction time of 0·7 s and ozone concentration of 0·08 and 0·25 mg l-1 , respectively, followed by tailing. Ozone disinfection kinetics of poliovirus 1 were better fit by the efficiency factor Hom model than by the Chick-Watson model, or the modified Chick-Watson model. Kinetics observed were similar between RT-qPCR and EMA-qPCR assays at the reaction times of <2·0 s and ozone concentrations of 0·08 and 0·25 mg l-1 . At reaction times > 5 s, viral concentration evaluated by EMA-qPCR was reduced in comparison to stable RT-qPCR results. Both assays still underestimated the virus inactivation. CONCLUSION: The simple developed reactor can be used to investigate viral ozone disinfection kinetics and to elucidate inactivation characteristics or mechanisms at very short exposure times. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed CQF reactor is beneficial for better understanding of virus inactivation by ozone, and the reactor can be used to better elucidate disinfection kinetics and mechanisms for future research. This work constitutes an important contribution to the existing knowledge of the application and limitation of the EMA/PMA-qPCR to assess virus infectivity after ozone disinfection.

Combination gemcitabine plus S-1 versus gemcitabine plus cisplatin for advanced/recurrent biliary tract cancer: the FUGA-BT (JCOG1113) randomized phase III clinical trial.

BACKGROUND: Gemcitabine plus cisplatin (GC) is the standard treatment of advanced biliary tract cancer (BTC); however, it causes nausea, vomiting, and anorexia, and requires hydration. Gemcitabine plus S-1 (GS) reportedly has equal to, or better, efficacy and an acceptable toxicity profile. We aimed to confirm the non-inferiority of GS to GC for patients with advanced/recurrent BTC in terms of overall survival (OS). PATIENTS AND METHODS: We undertook a phase III randomized trial in 33 institutions in Japan. Eligibility criteria included chemotherapy-naïve patients with recurrent or unresectable BTC, an Eastern Cooperative Oncology Group Performance Status of 0 - 1, and adequate organ function. The calculated sample size was 350 with a one-sided α of 5%, a power of 80%, and non-inferiority margin hazard ratio (HR) of 1.155. The primary end point was OS, while the secondary end points included progression-free survival (PFS), response rate (RR), adverse events (AEs), and clinically significant AEs defined as grade ≥2 fatigue, anorexia, nausea, vomiting, oral mucositis, or diarrhea. RESULTS: Between May 2013 and March 2016, 354 patients were enrolled. GS was found to be non-inferior to GC [median OS: 13.4 months with GC and 15.1 months with GS, HR, 0.945; 90% confidence interval (CI), 0.78-1.15; P = 0.046 for non-inferiority]. The median PFS was 5.8 months with GC and 6.8 months with GS (HR 0.86; 95% CI 0.70-1.07). The RR was 32.4% with GC and 29.8% with GS. Both treatments were generally well-tolerated. Clinically significant AEs were observed in 35.1% of patients in the GC arm and 29.9% in the GS arm. CONCLUSIONS: GS, which does not require hydration, should be considered a new, convenient standard of care option for patients with advanced/recurrent BTC. CLINICAL TRIAL NUMBER: This trial has been registered with the UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm), number UMIN000010667.

The State Scientific Automated Medical Registry, Kazakhstan: an important resource for low-dose radiation health research.

Direct quantitative assessment of health risks following exposure to ionizing radiation is based on findings from epidemiological studies. Populations affected by nuclear bomb testing are among those that allow such assessment. The population living around the former Soviet Union's Semipalatinsk nuclear test site is one of the largest human cohorts exposed to radiation from nuclear weapons tests. Following research that started in the 1960s, a registry that contains information on more than 300,000 individuals residing in the areas neighboring to the test site was established. Four nuclear weapons tests, conducted from 1949 to 1956, resulted in non-negligible radiation exposures to the public, corresponding up to approximately 300 mGy external dose. The registry contains relevant information about those who lived at the time of the testing as well as about their offspring, including biological material. An international group of scientists worked together within the research project SEMI-NUC funded by the European Union, and concluded that the registry provides a novel, mostly unexplored, and valuable resource for the assessment of the population risks associated with environmental radiation exposure. Suggestions for future studies and pathways on how to use the best dose assessment strategies have also been described in the project. Moreover, the registry could be used for research on other relevant public health topics.

Combined reconstructive surgery involving uterosacral colpopexy and anterior vaginal mesh implantation for pelvic organ prolapse.

AIM: The optimal treatment for pelvic organ prolapse has been the subject of much discussion. The aim of this study was to assess the utility of a combination of uterosacral colpopexy and anterior vaginal mesh implantation. METHODS: A single-center prospective cohort study was conducted. Twenty-eight patients with stage III-IV cystocele and uterine prolapse underwent reconstructive surgery. A combination of vaginal hysterectomy, McCall culdeplasty, and trocar-guided anterior vaginal mesh implantation was performed, and the patients' postoperative outcomes were analyzed. Patient satisfaction was investigated using the modified Short Form 12 version 2 (SF-12v2) questionnaire, and interviews regarding sexual behavior were conducted at 1 postoperative year. RESULTS: A bladder injury occurred during the dissection in one case (3.6%). Recurrent vaginal vault prolapse beyond the hymen was observed in one patient (cure rate: 96.4%), and further mesh augmentation was required in this case. Another patient developed mild cystocele (Ba = 0), but was simply observed because she did not complain of any symptoms caused by vaginal descent. We did not experience any other mesh-related complications, such as protrusion, chronic pain, or chronic inflammation, during the follow-up period. The patients' modified SF-12 scores at 12 months were significantly better than their preoperative scores in all eight domains. CONCLUSION: The satisfactory correction of pelvic organ prolapse was achieved using a combination of vaginal hysterectomy and uterosacral ligament colpopexy augmented by anterior vaginal mesh implantation. © 2016 Japan Society of Obstetrics and Gynecology.

Occurrence and reduction of human viruses, F-specific RNA coliphage genogroups and microbial indicators at a full-scale wastewater treatment plant in Japan.

AIMS: To evaluate and compare the reductions of human viruses and F-specific coliphages in a full-scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays. METHODS AND RESULTS: A total of 24 water samples were collected from four locations at the plant, and the relative abundance of human viruses and F-RNA phage genogroups were determined by qPCR. Of the 10 types of viruses tested, enteric adenoviruses were the most prevalent in both influent and effluent wastewater samples. Of the different treatment steps, the activated sludge process was most effective in reducing the microbial loads. Viruses and F-RNA phages showed variable reduction; among them, GI and GIII F-RNA phages showed the lowest and the highest reduction, respectively. CONCLUSIONS: Ten types of viruses were present in wastewater that is discharged into public water bodies after treatment. The variability in reduction for the different virus types demonstrates that selection of adequate viral indicators is important for evaluating the efficacy of wastewater treatment and ensuring the water safety. SIGNIFICANCE AND IMPACT OF THE STUDY: Our comprehensive analyses of the occurrence and reduction of viruses and indicators can contribute to the future establishment of appropriate viral indicators to evaluate the efficacy of wastewater treatment.

Circulating microRNAs (cmiRNAs) as novel potential biomarkers for hepatocellular carcinoma.

Incidence and mortality associated with hepatocellular carcinoma (HCC) is rising throughout the world. Accurate, noninvasive biomarkers for the early detection of HCC are urgently needed to reduce worldwide morbidity and mortality related to HCC. MicroRNAs (miRNAs), 17- to 25-nucleotide noncoding RNAs that are frequently dysregulated in HCC, have shown great promise as tissue-based markers for HCC diagnosis and prognosis. Moreover, they are stably expressed in serum and urine, and these circulating microRNAs (cmiRNAs) are emerging as novel noninvasive biomarkers for the early detection and prognosis of HCC. This article summarizes the latest findings on the role of circulating miRNAs as potential minimally invasive diagnostic and prognostic biomarkers for HCC.

Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles.

A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-A resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.

Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treatment plant in Japan.

AIMS: To investigate the prevalence, seasonality and genotype distribution of human noroviruses (NoVs) in wastewater in Japan. METHODS AND RESULTS: Influent and effluent water samples were collected monthly for a year from a wastewater treatment plant and examined for the presence of genogroups I and II (GI and GII) NoVs. Using real-time reverse transcription (RT)-PCR assays, 12 (100%) influent and six (50%) effluent samples were positive for both GI and GII NoV genomes, with a higher prevalence in winter. A total of 152 different NoV strains, comprising 84 GI and 68 GII strains, were identified using seminested RT-PCR assays followed by cloning and sequence analysis. These strains were classified into nine GI genotypes (GI/1, 2, 4, 5, 8, 9, 11, 12 and 14) and 13 GII genotypes (GII/1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15 and 16), showing considerable genetic diversity. CONCLUSIONS: Based on the partial capsid gene sequences, we identified a great number of NoV strains belonging to many genotypes, demonstrating that genetically diverse NoV strains are co-circulating in aquatic environments and human populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results clearly demonstrate the seasonal trend and genetic diversity of NoVs in wastewater.

Dose-response time modelling for highly pathogenic avian influenza A (H5N1) virus infection.

AIMS: To develop time-dependent dose-response models for highly pathogenic avian influenza A (HPAI) of the H5N1 subtype virus. METHODS AND RESULTS: A total of four candidate time-dependent dose-response models were fitted to four survival data sets for animals (mice or ferrets) exposed to graded doses of HPAI H5N1 virus using the maximum-likelihood estimation. A beta-Poisson dose-response model with the N(50) parameter modified by an exponential-inverse-power time dependency or an exponential dose-response model with the k parameter modified by an exponential-inverse time dependency provided a statistically adequate fit to the observed survival data. CONCLUSIONS: We have successfully developed the time-dependent dose-response models to describe the mortality of animals exposed to an HPAI H5N1 virus. The developed model describes the mortality over time and represents observed experimental responses accurately. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing time-dependent dose-response models for HPAI H5N1 virus. The developed models will be a useful tool for estimating the mortality of HPAI H5N1 virus, which may depend on time postexposure, for the preparation of a future influenza pandemic caused by this lethal virus.

Genetic diversity of genogroup IV noroviruses in wastewater in Japan.

AIMS: To investigate the prevalence, seasonality and genetic diversity of genogroup IV noroviruses (GIV NoVs) in wastewater in Japan. METHODS AND RESULTS: Untreated and treated wastewater samples were collected monthly for a year from a wastewater treatment plant in Japan. The concentrated wastewater samples were examined for the presence of GIV NoV genomes with seminested RT-PCR assay targeting partial capsid gene. Among 12 untreated and 12 treated wastewater samples tested, GIV NoV genomes were detected in three (25%) untreated and two (17%) treated wastewater samples with a high positive ratio in winter season. Genetic analysis revealed that the GIV NoVs in the wastewater samples were genetically diverse and were classified into three different genetic clusters. CONCLUSIONS: Frequent detection of GIV NoVs in winter season, which is a common epidemic period of human NoVs in Japan, indicates that GIV NoVs exhibit temporal trends similar to GI and GII NoVs. Based on the partial capsid gene sequences, we identified several unique GIV NoV strains belonging to the novel genetic cluster, demonstrating that GIV NoVs are more genetically diverse than previously appreciated. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings provide novel evidence of considerable genetic diversity among the GIV NoV strains.

Effect of photoreactivation on ultraviolet inactivation of Microcystis aeruginosa.

Microcystis aeruginosa forms algal bloom in lakes. They produce toxic compounds such as microcystin. Against such algal problems, the effect of UV treatment was examined. In UV treatment, the effect of photoreactivation should be examined. Photoreactivation is a repair mechanism of genomic DNA damage by sunlight irradiation. UV treatment causes DNA damages on target cyanobacteria, however sunlight can repair some of these DNA damages. To examine the effect of photoreactivation, both white and yellow light incubations were employed. White light allows both photoreactivation and photosynthesis, while yellow light prohibits photoreactivation and only allows photosynthesis. Microcystis aeruginosa NIES 98 strain and PCC 7806 strain were used as the test cultures. Those cultures were exposed to low-pressure (LP) or medium-pressure (MP) ultraviolet (UV) lamp, then incubated under white or yellow light. Yellow light incubation method was effective to examine photoreactivation. It was revealed that almost six times UV fluence was required to inactivate 99% of Microcystis aeruginosa, under photoreactivation condition, compared with non-photoreactivation condition. Inhibition of photoreactivation could greatly enhance UV treatment efficiency against Microcystis aeruginosa. One of the practical suggestions is to conduct UV treatment at night, when photoreactivation by sunlight rarely takes place. Highly efficient inactivation was achieved by avoiding photoreactivation.

Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure.

A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 µg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.

Crustacean hyperglycemic hormone (CHH) neuropeptidesfamily: Functions, titer, and binding to target tissues.

The removal of the eyestalk (s) induces molting and reproduction promoted the presence of regulatory substances in the eyestalk (ES), particularly medulla terminalis X-organ and the sinus gland (MTXO-SG). The PCR-based cloning strategies have allowed for isolating a great number of cDNAs sequences of crustacean hyperglycemic hormone (CHH) neuropeptides family from the eyestalk and non-eyestalk tissues, e.g., pericardial organs and fore- and hindguts. However, the translated corresponding neuropeptides in these tissues, their circulating concentrations, the mode of actions, and specific physiological functions have not been well described. The profiles of CHH neuropeptides present in the MTXO-SG may differ among decapod crustacean species, but they can be largely divided into two sub-groups on the basis of structural homology: (1) CHH and (2) molt-inhibiting hormone (MIH)/mandibular organ-inhibiting hormone (MOIH)/vitellogenesis/gonad-inhibiting hormone (V/GIH). CHH typically elevating the level of circulating glucose from animals under stressful conditions (hyper- and hypothermia, hypoxia, and low salinity) has multiple target tissues and functions such as ecdysteroidogenesis, osmoregulation, and vitellogenesis. Recently, MIH, known for exclusively suppressing ecdysteroidogenesis in Y-organs, is also reported to have an additional role in vitellogenesis of adult female crustacean species, suggesting that some CHH neuropeptides may acquire an extra regulatory role in reproduction at adult stage. This paper reviews the regulatory roles of CHH and MIH at the levels of specific functions, temporal and spatial expression, titers, their binding sites on the target tissues, and second messengers from two crab species: the blue crab, Callinectes sapidus, and the European green crab, Carcinus maenas. It further discusses the diverse regulatory roles of these neuropeptides and the functional plasticity of these neuropeptides in regard to life stage and species-specific physiology.

Chlorine inactivation of human norovirus, murine norovirus and poliovirus in drinking water.

AIMS: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. METHODS AND RESULTS: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench-scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0.1 and 0.5 mg l(-1). Inactivation of MNV reached more than 4 log(10) after 120 and 0.5 min contact time to chlorine at the initial free chlorine concentrations of 0.1 and 0.5 mg l(-1), respectively. CONCLUSIONS: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.

Detection of genogroup IV norovirus in wastewater and river water in Japan.

AIMS: To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV). METHODS AND RESULTS: Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption-elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6.9 x 10(4) and 1.5 x 10(4) copies l(-1), respectively. CONCLUSIONS: Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.

Large-scale copy number variants (CNVs) detected in different ethnic human populations.

The large-scale copy-number variations (CNVs) in the human genome are associated with developmental disorders and susceptibility to diseases. More importantly, CNVs may represent a major genetic component of our phenotypic diversity. Following the development of methodologies and introduction of new research platforms, accumulation of the nature and pattern of CNVs from normal populations has progressed. The examination of relatively large numbers of specific ethnic groups has recently started. Although the results are not always consistent, it is likely that different human populations bear different CNVs, as is the case for single-nucleotide polymorphisms (SNPs) and insertion-deletion (indel) polymorphisms. We review recent publications about the nature of inter-population, especially inter-ethnic group, differences of CNVs.

Prevalence of adult-onset multifactorial disease among offspring of atomic bomb survivors.

The first study to examine whether parental radiation exposure leads to increased heritable risk of common adult-onset multifactorial diseases (i.e., hypertension, diabetes mellitus, hypercholesterolemia, ischemic heart disease, and stroke) was conducted among 11,951 participants in the clinical examination program out of a potential of 24,673 mail survey subjects who were offspring of survivors born from May 1946 through December 1984. Logistic regression analyses demonstrated no evidence of an association between the prevalence of multifactorial diseases in the offspring and parental radiation exposure, after adjusting for age, city, gender and various risk factors. The odds ratio (OR) for a paternal dose of 1 Gy was 0.91 [95% confidence interval (CI) 0.81-1.01, P = 0.08], and that for a maternal dose of 1 Gy was 0.98 (95% CI 0.86-1.10, P = 0.71). There was no apparent effect of parental age at exposure or of elapsed time between parental exposure and birth, but male offspring had a low odds ratio (OR = 0.76 at 1 Gy) for paternal exposure, but cautious interpretation is needed for this finding. The clinical assessment of nearly 12,000 offspring of A-bomb survivors who have reached a median age of about 50 years provided no evidence for an increased prevalence of adult-onset multifactorial diseases in relation to parental radiation exposure.

Quantitative detection of sapoviruses in wastewater and river water in Japan.

AIMS: To detect sapoviruses at a wastewater treatment plant (WWTP) and in a river in Japan, quantitatively. METHODS AND RESULTS: Influent and effluent samples at a WWTP and river water samples were collected monthly for 1 year. The water samples were subjected to virus concentration using an HA electronegative filter, followed by quantification of sapoviruses using real-time PCR. The concentration of sapoviruses in influent ranged from 2.8 x 10(3) to 1.3 x 10(5) copies per litre, showing a higher value in winter. Seven (58%) of 12 effluent samples were positive for sapoviruses, as were 23 (64%) of 36 river water samples collected from three sites along the Tamagawa River. CONCLUSIONS: Sapoviruses were abundant in the influent even in the nonepidemic period, suggesting that sporadic and asymptomatic infections occur throughout the year. Increasing concentration of sapoviruses was discharged into the river during the epidemic period winter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study demonstrating the quantitative detection of sapoviruses in aquatic environments.

Induction of apoptosis in an estrogen-responsive mouse Leydig tumor cell by leukotriene.

For estrogen-responsive B-1F cells, established from estrogen-responsive mouse Leydig cell tumor, it has been reported that the 5-lipoxygenase (5-LOX) metabolic pathway appears to be associated with cell growth. The addition of 5-LOX inhibitor 2-(12-hydroxydodeca-5,10-diyl)-3,5,6-trimethyl-1,4-benzoquinone (AA861) to the medium resulted in a dose-dependent increase in cell yield as described previously. When the growth of the palpable tumors was measured, AA861 had stimulated in vivo tumor growth in adult male mouse inoculated B-1F cells. The effects of AA861 and 17beta-estradiol (E2) on the contents of various arachidonic acid metabolites in B-1F cells and their conditioned medium were examined. Although AA861 and E2 decreased the contents of leukotrienes (LTs), the two did not significantly change those of prostaglandins, thromboxan, prostacyclin, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE. In immunohistochemical study B-1F cells show positive staining for 5-LOX in the E2-depleted condition, while E2 decreased the expression of 5-LOX. The decrease of the intensities of 79-kDa 5-LOX protein and 403-bp RT-PCR product bands was observed. The growth of Morpholino-anti oligo delivered B-1F cells was higher than that of Standard control oligo delivered cells. The delivery of Morpholino-anti oligo into B-1F cells caused the decrease of contents of LTs and 5-HETE in the cells and medium, and the reduction of 5-LOX activity. When LTD4 was added in the culture medium, the increasing concentrations of LTD4 resulted in a significant inhibition of cell yields of E2-treated B-1F cells. Morphological changes such as nuclear condensation and fragmentation, and DNA ladder pattern were demonstrated in E2-stimulated B-1F cells treated with LTD4 as well as in control cells cultured in the basal medium. These results implicate that 5-LOX at least plays an important role in the growth of B-1F cells and LD4 induces the apoptosis of B-1F cells.