Koshidacins A and B, Antiplasmodial Cyclic Tetrapeptides from the Okinawan Fungus Pochonia boninensis FKR-0564.

Two new antiplasmodial peptides, named koshidacins A (1) and B (2), were discovered from the culture broth of the Okinawan fungus Pochonia boninensis FKR-0564. Their structures, including absolute configurations, were elucidated by a combination of spectroscopic methods and chemical derivatization. Both compounds showed moderate in vitro antiplasmodial activity against Plasmodium falciparum strains, with IC50 values ranging from 17.1 to 0.83 µM. In addition, compound 2 suppressed 41% of malaria parasites in vivo when administered intraperitoneally at a dose of 30 mg/kg/day for 4 days.

Clonocoprogens A, B and C, new antimalarial coprogens from the Okinawan fungus Clonostachys compactiuscula FKR-0021.

Three new antimalarial compounds, clonocoprogens A, B, and C, were isolated from the static culture of an Okinawan fungus, Clonostachys compactiuscula FKR-0021. These compounds were new analogs of N14-palmitoylcoprogen, reported as a siderophore. They showed moderate antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, with IC50 values ranging from 1.7 to 9.9 µM.

Corticotropin-Releasing Factor Receptor 1 in the Anterior Cingulate Cortex Mediates Maternal Absence-Induced Attenuation of Transport Response in Mouse Pups.

A human infant initially shows non-selective sociality, and gradually develops selective attachment toward its caregiver, manifested as "separation anxiety." It was unclear whether such sophistication of attachment system occurs in non-human mammals. To seek a mouse model of separation anxiety, we utilized a primitive attachment behavior, the Transport Response, in that both human and mouse newborns immediately stop crying and stay immobile to cooperate with maternal carrying. We examined the mouse Transport Response in three social contexts: 30-min isolation in a novel environment, 30-min maternal absence experienced with littermates in the home cage, and the control home-cage condition with the mother and littermates. The pups after postnatal day (PND) 13 attenuated their Transport Response not only in complete isolation but also by maternal absence, and activated several brain areas including the periventricular nucleus of the hypothalamus, suggesting that 30-min maternal absence was perceived as a social stress by mouse pups after PND13. This attenuation of Transport Response by maternal absence was independent with plasma corticosterone, but was diminished by prior administration of a corticotropin-releasing factor receptor 1 (CRFR1) antagonist. Among 18 brain areas examined, only neurons in the anterior cingulate cortex (ACC) co-express c-fos mRNA and CRFR1 after maternal absence. Consistently, excitotoxic ACC lesions inhibited the maternal absence-induced attenuation of Transport Response. These data indicate that the expression of mouse Transport Response is influenced not only by social isolation but also by maternal absence even in their home cage with littermates after PND13, at least partly via CRF-CRFR1 signaling in the ACC.

Determination of poor prognostic immune features of tumour microenvironment in non-smoking patients with lung adenocarcinoma.

We have previously demonstrated that the prognostic significance of tumour-infiltrating CD8+ T cells significantly differs according to histological type and patient smoking habits in non-small cell lung cancer (NSCLC). This work suggested that infiltrating CD8+ T cells may not be activated sufficiently in the immunosuppressive microenvironment in non-smokers with adenocarcinoma. To understand the immunogenic microenvironment in NSCLC, we characterised immune cells comprehensively by performing an immunohistochemical evaluation using an alternative counting method and multicolour staining method (n = 234), and assessed immune-related gene expression by using genetic analytical approaches (n = 58). We found that high infiltration of activated CD8+ T cells expressing interferon gamma (IFN-γ) and granzyme was correlated with postoperative survival in patients with non-adenocarcinoma. On the contrary, CD8+ T-cell accumulation was identified as a worse prognostic factor in patients with adenocarcinoma, particularly in non-smokers. Infiltrating CD8+ T cells were significantly less activated in this microenvironment with high expression of various immunoregulation genes. Potentially immunoregulatory CD8+ FOXP3+ T cells and immunodysfunctional CD8+ GATA3+ T cells were increased in adenocarcinoma of non-smokers. CD4+ FOXP3+ regulatory T cells expressing chemokine receptor-4 (CCR4)- and chemokine ligand (CCL17)-expressing CD163+ M2-like macrophages also accumulated correlatively and significantly in adenocarcinoma of non-smokers. These characteristic immune cells may promote tumour progression possibly by creating an immunosuppressive microenvironment in non-smoking patients with lung adenocarcinoma. Our findings may be helpful for refining the current strategy of personalised immunotherapy including immune-checkpoint blockade therapy for NSCLC.

Reference values for Japanese children's respiratory resistance using the LMS method.

BACKGROUND: The forced oscillation technique (FOT) is useful for studying pulmonary function in children, as well as in school children with asthma. However, the standard values for respiratory resistance (Rrs) in Asian school children remain unknown. We evaluated the standard Rrs using a type of FOT, impulse oscillometry (IOS), in healthy Japanese children at elementary and junior high schools. METHODS: A total of 795 children (age range: 6-15 years; mean age ± SD: 11.1 ± 2.4 years; 404 boys, 391 girls) at elementary and junior high schools participated in the study. Of the 795 children, we evaluated the Rrs of 537 children aged 6-15 years (mean ± SD: 10.8 ± 2.4 years) using IOS. RESULTS: Regression analyses with three IOS parameters, Rrs at 5Hz (R5), Rrs at 20Hz (R20), and Rrs difference between 5Hz and 20Hz (R5-R20), for age, height, weight, and degree of obesity as independent variables demonstrated the strongest correlation between each parameter and children's height. All parameters decreased with increasing height. Using the lambda-mu-sigma (LMS) method, we created standard curves for the Rrs values based on height. CONCLUSIONS: Our standard curves could be useful for diagnosis and control evaluation of childhood asthma.

Relationship between bronchial hyperresponsiveness and lung function in children age 5 and 6 with and without asthma.

BACKGROUND AND OBJECTIVE: It is unknown whether wheezy children have bronchial hyperresponsiveness (BHR) or which lung function parameters are correlated with BHR in children. We evaluated the relationship between BHR parameters and the lung functions by minimizing the effects of age and height in asthmatic, non-asthmatic wheezers and healthy children. METHODS: The subjects comprised of 154 children aged 5 and 6 years (78 males, 76 females), who were divided into three groups: asthmatics, non-asthmatic wheezers and healthy controls. Spirometry and a methacholine inhalation challenge by the oscillation method were performed. RESULTS: The age of the study cohort was 5.9 ± 0.2 years (mean ± standard deviation), and the height was 114.4 ± 5.3 cm. No significant differences in height, weight, body mass index or lung function were observed in the three groups. The minimal dose of methacholine to start bronchoconstriction, a parameter of bronchial sensitivity, was lower in asthmatics and non-asthmatic wheezers than that in controls. The speed of bronchoconstriction in response to methacholine, a parameter of bronchial reactivity, was strongly correlated with baseline respiratory resistance (Rrs cont) in all three groups. CONCLUSIONS: Our data suggest that it not possible to distinguish preschool children with asthma from non-asthmatic wheezers based on their bronchial sensitivity and that the baseline Rrs has a strong effect on the bronchial reactivity in children.

Tethering of proteins to RNAs using the bovine immunodeficiency virus-Tat peptide and BIV-TAR RNA.

To study the functions of RNA-binding proteins independent of their RNA-binding activity, tethering methods have been developed, based on the use of the RNA-binding domain of a well-characterized RNA-binding protein and its target RNA. Two bacteriophage proteins have mainly been used as tethers: the MS2 coat protein and the lambda N protein. Here we report an alternative system using the Tat (trans-activator) peptide from the bovine immunodeficiency virus (BIV), which binds to BIV-TAR (trans-activation response) RNA. We demonstrate the usefulness of this system by applying it to the analysis of the TNRC6B protein, a component of the microRNA-induced silencing complex.

Inducible protein expression in Drosophila Schneider 2 cells using the lac operator-repressor system.

Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO(4) or CdCl(2) as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator-repressor regulatory system functioned in chromosomally integrated cell lines.

Relationship between exhaled nitric oxide and small airway lung function in normal and asthmatic children.

BACKGROUND: In children, exhaled nitric oxide (eNO) is usually confounded by factors such as age and height. We evaluated the relationship between eNO and lung function by minimizing the effects of aging and height. METHODS: In Study 1, the subjects comprised 738 elementary school children and junior high school children (aged 6 to 15 years, 366 males and 372 females). They were divided into two groups according to age (6-10 years and 11-15 years). A height range was determined by a histogram of height in each group. In Study 2, lung function, respiratory resistance and eNO level were measured in age- and height-limited groups. RESULTS: In Study 1, total of 148 younger children ranging in height from 120 to 130 cm and 180 older children ranging in height from 148 to 158 cm participated in Study 2. The level of eNO among asthmatic children was higher than that of normal children in both the younger and the older groups. The decrease in forced expiratory volume in 1 second (FEV(1)) and other parameters of central airway resistance did not correlate with the eNO level. However, the small airway parameters of MMEF and V(25)/HT in older asthmatic children, and V(25)/HT and R(5)-R(20) in younger asthmatic children inversely correlated with eNO. CONCLUSIONS: Our data suggest that eNO level inversely correlates with small airway narrowing, and airway inflammation has a significant effect on small airway lung function in asthmatic school children.

Consistently high levels of exhaled nitric oxide in children with asthma.

BACKGROUND: Exhaled nitric oxide (eNO) levels in children are unstable because they are regulated by many potent factors. The purpose of the current study was to evaluate the reliability of eNO levels between a long interval and other lung functions in normal and asthmatic children. METHODS: Eighty-three elementary school children (aged 11-12 years; male : female, 39 : 44) participated in this study. Lung function, airway resistance and eNO levels were measured twice: the first measurement was in autumn 2007, and the second was one year later. RESULTS: There were 62 non-asthmatic control children (male : female, 31 : 31) and 21 asthmatic children (male : female, 8 : 13). In both the first and the second examination, the levels of eNO in children with asthma were higher than those in children without asthma. The parameters of lung function and the respiratory resistance in children without asthma showed a good correlation between the results of the first and second examinations. The eNO level in non-asthmatic children showed a good correlation between the two. On the other hand, the peripheral airway parameters of lung function and the respiratory resistance in children with asthma were not correlated between the first and the second examinations. The eNO level in these patients was well correlated between the two examinations. CONCLUSIONS: These data suggest that the eNO level showed good reproducibility in children with and without asthma. The eNO level is therefore considered to be a useful marker for reproducibly evaluating a subject's airway condition.

Changes in the highest frequency of breath sounds without wheezing during methacholine inhalation challenge in children.

BACKGROUND AND OBJECTIVE: It is difficult for clinicians to identify changes in breath sounds caused by bronchoconstriction when wheezing is not audible. A breath sound analyser can identify changes in the frequency of breath sounds caused by bronchoconstriction. The present study aimed to identify the changes in the frequency of breath sounds during bronchoconstriction and bronchodilatation using a breath sound analyser. METHODS: Thirty-six children (8.2 +/- 3.7 years; males : females, 22 : 14) underwent spirometry, methacholine inhalation challenge and breath sound analysis. Methacholine inhalation challenge was performed and baseline respiratory resistance, minimum dose of methacholine (bronchial sensitivity) and speed of bronchoconstriction in response to methacholine (Sm: bronchial reactivity) were calculated. The highest frequency of inspiratory breath sounds (HFI), the highest frequency of expiratory breath sounds (HFE) and the percentage change in HFI and HFE were determined. The HFI and HFE were compared before methacholine inhalation (pre-HFI and pre-HFE), when respiratory resistance reached double the baseline value (max HFI and max HFE), and after bronchodilator inhalation (post-HFI and post-HFE). RESULTS: Breath sounds increased during methacholine-induced bronchoconstriction. Max HFI was significantly greater than pre-HFI (P < 0.001), and decreased to the basal level after bronchodilator inhalation. Post-HFI was significantly lower than max HFI (P < 0.001). HFI and HFE were also significantly changed (P < 0.001). The percentage change in HFI showed a significant correlation with the speed of bronchoconstriction in response to methacholine (P = 0.007). CONCLUSIONS: Methacholine-induced bronchoconstriction significantly increased HFI, and the increase in HFI was correlated with bronchial reactivity.

Relationship between bronchial hyperreactivity and asthma remission during adolescence.

BACKGROUND: Many children with asthma outgrow this disease after the onset of puberty. However, the precise mechanism of outgrowing asthma in children is still unclear. OBJECTIVE: To evaluate the characteristics of respiratory physiology during adolescence. METHODS: The results of the lung function test and methacholine inhalation challenge were prospectively evaluated in adolescent patients with asthma with and without symptoms. One hundred sixty children with asthma participated. Twenty-eight children had symptom-free adolescent asthma (i.e., remission asthma) (boy to girl ratio, 16:12; mean age, 14.6 years), 25 had intermittent adolescent asthma (boy to girl ratio, 16:9; mean age, 14.9 years), and 47 had symptomatic adolescent asthma (boy to girl ratio, 27:20; mean age, 12.7 years). For comparison purposes, 60 younger children with symptomatic asthma participated. The parameters of bronchial hyperresponsiveness, baseline respiratory resistance, threshold of methacholine (Dmin) (bronchial sensitivity), and speed of bronchial constriction (Sm) (bronchial reactivity) were measured by methacholine inhalation challenge using the continuous oscillation method. RESULTS: There was no significant difference in lung function results, such as forced vital capacity and forced expiratory volume in 1 second, between the intermittent asthma and the remission asthma groups. Also, there was no significant difference in baseline respiratory resistance and Dmin between the 2 groups. However, the value of Sm of the remission asthma group was significantly lower than that of the intermittent asthma group (P = .02) and the symptomatic asthma group (P = .02). CONCLUSIONS: These data show that the adolescents with asthma remission showed a significant decrease of Sm, whereas Dmin was not changed. These results suggest one of the mechanisms by which asthma is outgrown in children and explain the common clinical aspects of adolescent asthma, such as symptom-free but bronchial hyperresponsive asthma.

Effects of skin care with shower therapy on children with atopic dermatitis in elementary schools.

For elementary school children with atopic dermatitis, a skin care program using shower therapy was performed during the school lunch break for 6 weeks from June to July in 2004 and 2005. All 53 participants showed an improvement in their atopic dermatitis during the 6-week periods studied. Skin care with daily showering at an elementary school was thus found to be effective for the treatment of atopic dermatitis.

SMG-8 and SMG-9, two novel subunits of the SMG-1 complex, regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed "SURF" that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.

Effect of aging on the relationship between asthma severity and bronchial hyperresponsiveness in children with asthma.

An association between asthma and bronchial hyperresponsiveness (BHR) has been demonstrated. It is possible that the relationship between asthma severity and BHR in children with asthma is different in infants and in adolescents. The aim of this study is therefore to evaluate the effect of aging on the relationship between the severity of asthma and BHR in children with asthma. We measured BHR in 386 subjects ranging from 2 to 20 years of age. The subjects consisted of 323 children with asthma (boys:girls = 193:130, mean age 9.7 years) and 63 age-matched controls (boys:girls = 25:38, mean age 8.2 years). BHR was measured using the methacholine inhalation challenge by measuring the transcutaneous oxygen pressure (tcPO2) in children less than 6 years of age (Dmin-PO2) and by measuring the respiratory resistance (Rrs) in children 6 years of age and older (Dmin-Rrs). Throughout the whole age range, both the Dmin-PO2 and Dmin-Rrs in each asthma severity group were higher than those in the controls. In the asthmatics aged 2-5 years, the Dmin-PO2 levels in the mild asthma group were higher than those in the moderate and severe asthma groups (p < 0.001, p < 0.001, respectively), and the Dmin-PO2 levels in the moderate asthma group were also higher than those in the severe asthma group. This tendency was also found in the age ranges of 6-9 years and 10-13 years. In the asthmatics aged 14-20 years, the Dmin-Rrs levels were not significantly different among the three groups. Taken together, these data show that aging has an effect on the relationship between the severity of asthma and BHR during childhood and that BHR may not be the sole determinant for the severity of asthma in adolescence.

Effect of inhaled histamine on airway epithelial cell swelling in ozone-exposed Guinea pigs.

BACKGROUND: It has been reported that histamine stimulates ion channels on airway epithelial cells and induces changes in osmolarity and the ion composition of the periciliary field of airway epithelia. OBJECTIVE: To investigate the effect of inhaled histamine on epithelial cell swelling, we studied the role of airway epithelial cells under histamine inhalation challenge in an animal model of airway inflammation using ozone exposure. METHOD: After exposure to 3.0 ppm ozone for 2 h, guinea pigs were anesthetized and tracheostomized, and then, lung resistance (R(L)) was measured. Histamine inhalation challenge and histological examination were performed. RESULT: The values of R(L) before histamine inhalation in the control group and the ozone-exposed group were 0.26 +/- 0.11 and 0.45 +/- 0.34 cm H(2)O/s, respectively. R(L) increased significantly after histamine inhalation both in the control and the ozone-exposed groups. The threshold of histamine (PC(200)) in the ozone-exposed group was significantly lower than that in the control group. A significant swelling of the epithelial cells after histamine inhalation was observed both in the control and the ozone-exposed groups, with a greater increase in the ozone-exposed group compared with the control group. However, no change in wall thickness was observed in the histamine/antihistamine or the ozone/histamine/antihistamine group. CONCLUSION: Our results suggest the possibility that the airway epithelial cell swelling plays a role in the increase in R(L) after histamine inhalation, especially in the presence of airway inflammation.

Evaluation of bronchial hyperresponsiveness by monitoring of transcutaneous oxygen tension and arterial oxygen saturation during methacholine challenge in asthmatic children.

BACKGROUND: Bronchial hyperresponsiveness (BHR) is a key feature of asthma, but the measurement of BHR is hampered by the fact that most tests of airway caliber are difficult to conduct at a young age. Methacholine-induced bronchoconstriction is associated with significant hypoxemia, which can be assessed noninvasively by transcutaneous oxygen pressure (tcPO2) and pulse oximetry. Evaluating BHR by monitoring tcPO2 instead of respiratory resistance (Rrs) has been used over a wide age range in childhood. OBJECTIVE: To investigate whether there is a consistent relationship between changes in arterial oxygen saturation (SaO2) and respiratory resistance (Rrs) similar to the relationship between tcPO2 and Rrs during methacholine challenge in young children and to assess the usefulness of SaO2 as a parameter for the indirect measurement of BHR. METHOD: We performed methacholine inhalation challenge by monitoring SaO2, tcPO2 and Rrs in 37 asthmatic children 5 to 7 years of age. Consecutive doses of methacholine were doubled until a 10% decrease in tcPO2 from the baseline was reached. We recorded the cumulative dose of methacholine (Dmin) at the inflection point of tcPO2 (Dmin-tcPO2), SaO2 (Dmin-SaO2), and Rrs(Dmin-Rrs). RESULTS: The mean value of Dmin-Rrs was 4.27 +/- 2.02 units, the mean value of Dmin-tcPO2 was 4.48 +/- 2.01 units, and the mean value of Dmin-SaO2 was 4.57 +/- 0.20 units. Inhalation of increasing doses of methacholine raised Rrs curvilinearly and depressed tcPO2 and SaO2. There were no significant differences between any of the parameters. There were significant relationships between Dmin-tcPO2 and Dmin-Rrs (r = 0.914, p < 0.001) and between Dmin-SaO2 and Dmin-Rrs (r = 0.905, p < 0.001) and a relationship between Dmin-tcPO2 and Dmin-SaO2 (r = 0.949, p < 0.001). CONCLUSION: We concluded that measurement of SaO2 and/or tcPO2 during methacholine inhalation challenge may be used to assess bronchial hyperresponsiveness. This study showed that both SaO2 and tcPO2 monitoring are safe, useful, and tolerable for use in children who are too young to cooperate with lung function tests.