Effect of miR-433-3p and miR-883b-5p on murine CYP 3A family enzymes in AML12 cells.

Here we searched for microRNAs that could interact with cytochrome P450 (CYP) enzymes in silico, and then investigated their effects on Cyp gene expressions using the cultured mouse liver cell line AML12. Among the mouse Cyp3a genes, some miRNAs were found to interact with Cyp3a11, 13, 16, and 44 by the in silico analysis using the miRWalk2.0 database. In addition to this software, which included twelve miRNA target prediction algorithms, we also applied our in-house-developed Excel VBA algorithm to obtain predictions more efficiently. Finally, two miRNAs, miR-433-3p and miR-883b-5p, were extracted as candidates that interact with Cyp3a genes. To evaluate the effects of these miRNAs on Cyp3a gene expression, we first examined whether they actually interacted with the Cyp3a 3'-untranslated region (3'-UTR) using a luciferase assay system in AML12 cells. We then evaluated whether the expression of each miRNA affected the expression of Cyp3a mRNAs and their transcribed proteins. We found that the transiently expressed miRNAs significantly reduced the reporter activity of the Cyp3a 3'-UTR site in AML12 cells. In addition, the mRNA and protein expressions of the corresponding Cyp3as were significantly decreased in the miRNA-treated AML12 cells. Using cultured cells, we clearly demonstrated that miR-433-3p and miR-883b-5p, which were identified by in silico prediction, actually bind to Cyp3a mRNAs and regulate Cyp gene expressions.

Identification of patterns of factors preceding severe or life-threatening asthma exacerbations in a nationwide study.

BACKGROUND: Reducing near-fatal asthma exacerbations is a critical problem in asthma management. OBJECTIVES: To determine patterns of factors preceding asthma exacerbations in a real-world setting. METHODS: In a nationwide prospective study of 190 patients who had experienced near-fatal asthma exacerbation, cluster analysis was performed using asthma symptoms over the 2-week period before admission. RESULTS: Three distinct clusters of symptoms were defined employing the self-reporting of a visual analogue scale. Cluster A (42.1%): rapid worsening within 7.4 hours from moderate attack to admission, young to middle-aged patients with low Body mass index and tendency to depression who had stopped anti-asthma medications, smoked, and hypersensitive to environmental triggers and furred pets. Cluster B (40.0%): fairly rapid worsening within 48 hours, mostly middle-aged and older, relatively good inhaled corticosteroid (ICS) or ICS/long-acting beta-agonist (LABA) compliance, and low perception of dyspnea. Cluster C (17.9%): slow worsening over 10 days before admission, high perception of dyspnea, smokers, and chronic daily mild-moderate symptoms. There were no differences in overuse of short-acting beta-agonists, baseline asthma severity, or outcomes after admission for patients in these 3 clusters. CONCLUSION: To reduce severe or life-threatening asthma exacerbation, personalized asthma management plans should be considered for each cluster. Improvement of ICS and ICS/LABA compliance and cessation of smoking are important in cluster A. To compensate for low perception of dyspnea, asthma monitoring of peak expiratory flow rate and/or exhaled nitric oxide would be useful for patients in cluster B. Avoidance of environmental triggers, increase usual therapy, or new anti-type 2 response-targeted therapies should be considered for cluster C.

Severe or life-threatening asthma exacerbation: patient heterogeneity identified by cluster analysis.

BACKGROUND: Severe or life-threatening asthma exacerbation is one of the worst outcomes of asthma because of the risk of death. To date, few studies have explored the potential heterogeneity of this condition. OBJECTIVES: To examine the clinical characteristics and heterogeneity of patients with severe or life-threatening asthma exacerbation. METHODS: This was a multicentre, prospective study of patients with severe or life-threatening asthma exacerbation and pulse oxygen saturation < 90% who were admitted to 17 institutions across Japan. Cluster analysis was performed using variables from patient- and physician-orientated structured questionnaires. RESULTS: Analysis of data from 175 patients with severe or life-threatening asthma exacerbation revealed five distinct clusters. Cluster 1 (n = 27) was younger-onset asthma with severe symptoms at baseline, including limitation of activities, a higher frequency of treatment with oral corticosteroids and short-acting beta-agonists, and a higher frequency of asthma hospitalizations in the past year. Cluster 2 (n = 35) was predominantly composed of elderly females, with the highest frequency of comorbid, chronic hyperplastic rhinosinusitis/nasal polyposis, and a long disease duration. Cluster 3 (n = 40) was allergic asthma without inhaled corticosteroid use at baseline. Patients in this cluster had a higher frequency of atopy, including allergic rhinitis and furred pet hypersensitivity, and a better prognosis during hospitalization compared with the other clusters. Cluster 4 (n = 34) was characterized by elderly males with concomitant chronic obstructive pulmonary disease (COPD). Although cluster 5 (n = 39) had very mild symptoms at baseline according to the patient questionnaires, 41% had previously been hospitalized for asthma. CONCLUSIONS & CLINICAL RELEVANCE: This study demonstrated that significant heterogeneity exists among patients with severe or life-threatening asthma exacerbation. Differences were observed in the severity of asthma symptoms and use of inhaled corticosteroids at baseline, and the presence of comorbid COPD. These findings may contribute to a deeper understanding and better management of this patient population.

Three-dimensional characterization of ODS ferritic steel using by FIB-SEM serial sectioning method.

Considerable attention has been paid to the research of the electron tomography due to determine the three-dimensional (3D) structure of materials [1]. One of the electron tomography techniques, focused ion beam/scanning electron microscopy (FIB-SEM) imaging has advantages of high resolutions (10 nm), large area observation (µm order) and simultaneous energy dispersive x- ray microanalysis (EDS)/ electron backscatter diffraction (EBSD) analysis. The purpose of this study, three-dimensional EBSD analysis of ODS ferritic steel which carried out cold work using FIB-SEM equipment was conducted, and it aimed at analyzing the microstructure obtained there. The zone annealing tests were conducted for ferritic steel [2,3], which were produced through mechanical alloying and hot-extrusion. After zone annealing, specimens were mechanically polished with #400∼4000 emery paper, 1 µm diamond paste and alumina colloidal silica. The serial sectioning and the 3D-electron backscattering diffraction (3D-EBSD) analysis were carried out. We made the micro pillar (30 x 30 x 15 µm). The EBSD measurements were carried out in each layer after serial sectioning at a step size and milling depth was 80 nm with 30 slices. After EBSD analysis, the series of cross-sectional images were aligned according to arbitrarily specified areas and then stacked up to form a volume. Consequently, we obtained the 3D-IPF maps for ODS ferritic steel. In this specimen, the {111} and {001} grains are layered by turns. In addition, the volume fraction value of both plane are similar. The aspect ratio increases with specimen depth. The 3D-EBSD mapping is useful to analysis of the bulk material since this method obtain many microstructure information, such a shape, volume and orientation of the crystal, grain boundary.

Serum lipid levels are positively associated with non-erosive reflux disease, but not with functional heartburn.

BACKGROUND: Metabolic syndrome and obesity are known risk factors for gastro-esophageal reflux disease (GERD), especially for erosive esophagitis. Although non-erosive reflux disease (NERD) is probably associated with obesity or other metabolic syndrome, there is little direct evidence to support this assertion. METHODS: Workers in Keio University who underwent a general health examination between September 2006 and August 2007 were enrolled. Reflux symptom questionnaires were administered and metabolic parameters were obtained. The severity of gastro-esophageal reflux (GER) was scored using a validated scale of videoesophagography. KEY RESULTS: Two hundred and eighty-three subjects (243 men and 40 women; mean age 49.8 +/- 6.9 years) with no radiographic evidence of erosive esophagitis were enrolled. The severity of GER was worse among men than among women, whereas the severity of reflux symptoms was worse among women. The severity of GER was associated with age and serum triglyceride levels in men, and with the serum low-density lipoprotein cholesterol levels in women. The severity of reflux symptoms, however, was not associated with metabolic parameters. There were more women than men with reflux symptoms but without GER ('presumed' functional heartburn group), compared with subjects with neither GER nor reflux symptoms. In men, the presence of both reflux symptoms and GER ('presumed' NERD group) was associated with the serum triglyceride levels. CONCLUSIONS & INFERENCES: While NERD is associated with serum lipid levels, functional heartburn is not. The prevalence of GER was greater among men; conversely, the prevalence of functional heartburn was greater among women.

Voided stain on paper method for analysis of mouse urination.

AIMS: To evaluate the usefulness of a quantification method using filter paper for analyzing minute voided urine of the mouse. METHODS: Voided stain on paper (VSOP) method; the correlation between area of stained spot on a filter paper and amount of applied liquid was calculated. Voiding behavior of the mice was analyzed by placing the animal above the same filter paper and recording voided time and area over 2 hr. The usefulness of the VSOP method was tested in analysis of the voiding behavior of five female 7-week-old ddY mice treated with cyclophosphamide (CPM, 150 mg/kg, intraperitoneally) and five control ones, in comparison with the histology of CPM-induced cystitis. Further, the voided volume of male and female ddY mouse ranging from 2 to 13 weeks was assessed. RESULTS: There was a linear correlation between liquid volume and stained area on the filter paper (y = 16.472x - 22.411, R(2) = 0.9981). Between control mice and those with histologically proven CPM cystitis, there was a significant difference in voided volume (362.7 +/- 51.9 and 127.8 +/- 100.0 microl, < 0.001) and voiding interval (10.30 +/- 3.10 and 4.47 +/- 1.70 min, < 0.001). Voided volume of ddY mice was quantifiable from as early as 2-week old, increased along with their growth and correlated well with their body weight [(voided volume: microl) = 10.8 x (body weight: g) + 32, R(2) = 0.762]. CONCLUSIONS: The VSOP method is a useful tool for evaluating voiding behavior of the mouse, including those with small bladder capacity.

Epigenetic silencing of prostaglandin E receptor 2 (PTGER2) is associated with progression of neuroblastomas.

We previously identified a cluster of prostanoid receptor genes, prostaglandin D2 receptor (PTGDR) and prostaglandin E receptor 2 (PTGER2), as possible targets for DNA methylation in advanced types of neuroblastoma (NB) using bacterial artificial chromosome array-based methylated CpG island amplification method. Among them, in this study, we found that PTGER2 was frequently silenced in NB cell lines, especially in those with MYCN amplification, through epigenetic mechanisms. In NB cell lines, DNA methylation pattern within a part of CpG island was inversely correlated with PTGER2 expression, and histone H3 and H4 deacetylation and histone H3 lysine 9 methylation within the putative promoter region were more directly correlated with silencing of this gene. Methylation of PTGER2 was observed more frequently in advanced-type of primary NBs compared with early-stage tumors. Growth of NB cells lacking endogenous PTGER2 expression was inhibited by restoration of the gene product by transient and stable transfection. A PTGER2-selective agonist, butaprost, increased intracellular cyclic adenosine monophosphate (cAMP) level, inhibited cell growth and induced apoptosis of NB cells stably expressing exogenous PTGER2. 8-Bromo-cAMP also inhibited growth of NB cells lacking PTGER2 expression, but not cells expressing this gene. Taken together, it is suggested that NB cells may lose responsiveness to PTGER2-mediated growth inhibition/apoptosis through epigenetic silencing of PTGER2 and/or disruption of downstream cAMP-dependent pathway during the neuroblastomagenesis.

Genotyping of congenital adrenal hyperplasia due to 21-hydroxylase deficiency presenting as male infertility: case report and literature review.

We describe here two infertile male patients who were referred to our hospital with azoospermia at the ages of 33 and 30 years, respectively. Hormonal examinations led to a diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency in both patients. Genotyping revealed that the patients had a homozygous I172N and a heterozygous compound I172N/IVS2-13A/C>G mutation, respectively. Glucocorticoid replacement therapy succeeded in improving the seminal status of one patient, but not the other. For the latter patient and his wife, a pregnancy was achieved by testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) following genetic counseling. It is important to investigate genotyping and to classify patients on the basis of genotypic information in order to arrive at better treatment strategies for male infertility; especially in counseling of TESE-ICSI.

Chorioretinal temperature monitoring during transpupillary thermotherapy for choroidal neovascularisation.

AIMS: To investigate the difference in temperature rise between normal choroid and choroidal revascularisation (CNV) during transpupillary thermotherapy (TTT) and the relation between laser spot size and power in the rat fundus. METHODS: A modified slit lamp, which was installed with two laser wavelengths (490 nm for illumination and fluorescein excitation and 810 nm for hyperthermia), was developed for TTT and temperature monitoring. Temperature rise during TTT was monitored by observing fluorescence released from thermosensitive liposomes encapsulating carboxyfluorescein. Two types of liposomes were prepared; their phase transition temperatures were 40 degrees C and 46 degrees C, respectively. Laser power settings required to observe fluorescence released from 46 degrees C liposome in normal choroid or CNV were compared. Next, the power settings with 0.5 mm and 0.25 mm spot sizes were compared following administration of 40 degrees C liposome or 46 degrees C liposome. RESULTS: The minimum power values when release from 46 degrees C liposome was observed showed a significant difference in distribution of power values between normal choroid and CNV. CNV required significantly higher power than normal choroid. With 40 degrees C liposome, the power was 9.7 (1.9) mW (mean (SD)) at a spot size of 0.25 mm, and 12.1 (1.6) mW at 0.5 mm, respectively. When using 46 degrees C liposome, the power setting was 10.2 (1.2) mW at a spot size of 0.25 mm, and 14.6 (2.2) mW at 0.5 mm, respectively. CONCLUSIONS: CNV demonstrated varying heat conduction, compared with normal choroid. Laser power required to raise the temperature should not necessarily be doubled, even when the spot size is doubled. Close attention should be given to the selection of power settings when performing TTT for CNV.

[Antimicrobial activities and mechanisms of carbapenem resistance in clinical isolates of carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter spp].

We tested the antimicrobial activities of meropenem (MEPM), imipenem (IPM), panipenem (PAPM), piperacillin (PIPC), cefepime (CFPM), aztreonam (AZT), amikacin (AMK), and levofloxacin (LVFX) against 106 clinical Pseudomonas aeruginosa isolates and 64 clinical Acinetobacter spp. isolates with reduced susceptibility to carbapenems. Using NCCLS breakpoints, the percentages of P. aeruginosa strains susceptible to AMK and Acinetobacter spp. strains susceptible to LVFX were found to be 51.1% and 55.6%, respectively, which represented the highest activity among 8 antimicrobial agents in each organism. Referring to the correlations among MICs of carbapenems, MEPM showed a higher activity than IPM and PAPM in both organisms; 29 of the 94 strains (30.9%) of IPM-resistant P. aeruginosa were susceptible to MEPM. Further study for resistance mechanisms to carbapenems by the disk diffusion method using 2-mercaptopropionic acid revealed that 8 of the 64 Acinetobacter spp. isolates (12.5%) were metallo-beta-lactamase producers, while none of 106 P. aeruginosa isolates were metallo-beta-lactamase producers. PCR analysis using blaIMP-specific primers confirmed that 4 of the 8 metallo-beta-lactamase-producing Acinetobacter spp. isolates detected by the disk diffusion method were carrying the blaIMP gene. The identification of metallo-beta-lactamase-producing Acinetobacter spp. isolates implies that metallo-beta-lactamase genes have been disseminated among various gram-negative pathogens.

[Malignant fibrous histiocytoma originating in a renal capsule: a case report].

We report a case of malignant fibrous histiocytoma originating in a renal capsule. A 43-year-old woman was admitted with a chief complaint of right lower abdominal pain. Physical examination was unremarkable. Serum C-reactive protein and erythrocyte sedimentation rate increased to 3.8 mg/dl and 60 mm/hr., respectively. Computed tomography (CT) and magnetic resonance image (MRI) showed a heterogeneous enhanced mass, 4 x 9 x 13 cm in size, in contact with the lateral part of the right kidney. Selective right renal arteriography revealed a hypovascular tumor, the main feeding artery of which was the right adrenal artery. Preoperative clinical diagnosis was a retroperitoneal sarcoma and transabdominal tumor resection was performed. The adhesion between the tumor and the right kidney was so severe that right nephrectomy was also necessary for a radical surgery. The tumor, measuring 13 x 9 x 6 cm, was located laterally adhering to the right renal capsule. Microscopic examination of the tumor demonstrated spindle-shaped fibroblast-like cells arranged in a storiform pattern with fibrous stroma and clusters of rounded histiocyte-like cells and pleomorphic giant cells with bizarre nuclei. Histopathological diagnosis was malignant fibrous histiocytoma arising from the renal capsule and there was no tumor invasion to renal parenchyma. No adjuvant therapy was performed but she has remained well for 31 months since the operation without evidence of disease.

In vivo development of decreased susceptibility to vancomycin in clinical isolates of methicillin-resistant Staphylococcus aureus.

To investigate the possibility of in vivo development of decreased vancomycin susceptibility, the vancomycin susceptibilities of 12 methicillin-resistant Staphylococcus aureus (MRSA) isolates serially recovered from six patients with vancomycin therapy were tested by standard MIC determination method and population analysis. While all of the MRSA isolates were susceptible to vancomycin (MICs, 1-2 microg/ml) by standard method, population analysis showed the upward shifts indicating decreased vancomycin susceptibility among serial isolates from two patients. These bacteria with decreased vancomycin susceptibility could be selected by using vancomycin selection of pre-therapy isolates under laboratory conditions. Furthermore, the reversion phenomenon of decreased vancomycin susceptibility was confirmed after 20 serial passages of the post-therapy isolates on drug-free agar. These data suggest that in vivo isolates may develop decreased vancomycin susceptibility that is not of such magnitude to cross a breakpoint threshold. This resistance may be unstable, and appears to result from a selective or inducible process that occurs in MRSA clinical strains during vancomycin therapy.

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity.

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.

Generation of new transposons in vivo: an evolutionary role for the "staggered" head-to-head dimer and one-ended transposition.

From a plasmid carrying the tnpA gene and one inverted repeat sequence (IR) of transposon Tn3, plasmids containing a structure characteristic of transposons, i.e., two IRs flanking a tnpA gene, were generated spontaneously in vivo. They appear to have arisen either through the formation of a "staggered" head-to-head dimer or by so-called one-ended transposition. These putative transposons could indeed transpose to, or form cointegrates with, a recipient plasmid. Based on these findings it is proposed that a primeval transposase gene and its target site evolved first, and subsequently gave rise to a "fully-fledged" transposon by head-to-head dimerization or one-ended transposition.

Cell adhesion in the process of asexual reproduction of tunicates.

Cell adhesion during budding of tunicates is reviewed from the viewpoints of histology, cytology, biochemistry, and molecular biology. Two kinds of multipotent cells play important roles in bud formation and development: epithelial cells, such as the atrial epithelium of botryllids and polystyelids, and mesenchymal cells, referred to as haemoblasts. Haemoblasts are able to aggregate to form a solid mass of cells, which soon becomes a hollow vesicle. The vesicular epithelium has junctional complexes that contain adherens junctions, and, sometimes, tight junctions; both occur apicolaterally on the plasma membrane. The hollow vesicle develops into the heart, the pyloric gland and duct, the gonad, including germ cells, and even the multipotent epithelium of buds. Cell culture studies suggest that multipotent epithelial cells may be interchangeable with haemoblasts. Several kinds of calcium-dependent, galactose-binding tunicate lectins (TC-14s) have been isolated and sequenced, and have been found to facilitate both in vivo and in vitro cell aggregation and migration. Tunicate homologs of cadherin and integrin genes have recently been isolated from Botryllus and Polyandrocarpa, respectively. Their unique molecular characteristics are discussed in the context of roles that they play in cell adhesion in the process of tunicate budding.

[Radiologic features of superficial-type hyperplastic polyps in the large intestine].

We studied 38 superficial-type hyperplastic polyps of more than 0.6 cm in size on radiographs to examine their morphologic features. The 38 lesions ranged in size from 0.6 cm to 2.5 cm, and 22 were located in the sigmoid and transverse colon (Table 1, Fig.1). The cross-section and surface configurations of the 38 lesions studied radiographically are illustrated in Fig. 2. 24 lesions without central depression were classified into 5 groups (1-a to 1-e). The 14 other lesions had central depression (2-a). The cross-section configuration was sharp in all lesions, and a finely serrated arrangement was seen in 30 of them. In surface configuration, several granular patterns were seen, but full-blown undulated folds and/or regular granular patterns were characteristic in groups 1-c, 1-d and 2-a. These radiologic features of cross-section and surface configuration may be regarded as a possible means of distinguishing superficial-type hyperplastic polyps from adenomas and carcinomas of the large intestine.

The molecular basis of the instability of a crp- mutation in Escherichia coli.

We have described a rapid spontaneous conversion in the stationary phase of Escherichia coli strain DOO (crp-) cells as a whole population to crp+ state (Sugino and Morita, 1994). In this paper we have tried to elucidate the molecular basis of this unidirectional conversion by cloning and sequencing of the crp gene in their crp+ and crp- states. We have found that in the original crp- strain, an IS2 element has been inserted between its original promoter and the coding region of the crp gene in the so-called orientation II (Ahmed et al., 1981), accompanied by an 11 bp deletion. Unexpectedly, the crp+ "revertants" derived from the crp- mutant had no difference in sequence from the crp-, either in the coding or the regulatory region. This suggests that a change at another locus, such that this change somehow activates the expression of the crp gene to the level of a normal crp+, is responsible for the apparent reversion from crp- to crp+.

[Three cases of thymic hyperplasia associated with hyperthyroidism].

We encountered three cases of thymic hyperplasia associated with hyperthyroidism. Case 1 was in a 35-year-old woman; a chest CT scan showed an anterior mediastinal mass and right-sided pleural effusion, which suggested the presence of a thymoma Case 2 was in a 21-year-old man who complained of palpebral ptosis and also had myasthenia gravis (Osserman type I). Case 3 was in a 47-year-old woman; a chest CT scan showed thymic hyperplasia and mediastinal lymphadenopathy. In all cases, anti-thyroid medication was given first, because of the associations with hyperthyroidism. Moreover, in cases 1 and 2 no tumor was found, and only hyperplasia was detected in the thymus, although both patients underwent extended thymectomy. Furthermore, surgery was not effective against the hyperthyroidism (anti-thyroid medication could not be withdrawn or reduced). In cases 2 and 3, thymic hyperplasia, as seen on chest CT scans, resolved as thyroid function was normalized by anti-thyroid medication. The pretracheal lymphadenopathy seen in case 3 also resolved. Thymic hyperplasia may have been a result, not a cause, of hyperthyroidism. When we encounter patients with thymic masses and hyperthyroidism, we should give anti-thyroid medication and observe the thymus for some time before resorting to surgery.

Nested deletions from a fixed site as an aid to nucleotide sequencing: an in vitro system using Tn3 transposase.

We have previously constructed a cloning/sequencing vector, with an in vivo system capable of creating nested deletions from the end of transposon Tn3, which is useful for sequencing large DNAs. Here we report an in vitro system which uses an ammonium sulfate fraction of extract from E. coli cells harboring a Tn3 transposase overproducer plasmid to generate nested deletions. A key feature of the procedure is exhaustive digestion of the reaction products with a restriction enzyme that cleaves only between the Tn3 "right" terminus and the cloned fragment. This step reduces the noise level due to mechanisms other than deletions from the Tn3 terminus, and facilitates detection and isolation of the desired deletion products. This system enables us to save at least 2 days' time when obtaining the necessary deletions compared with the in vivo system.