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.

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.

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.

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.

Tumor angiogenesis activity in clonal cells transformed by bovine adenovirus type 3.

Four different sets of clonal cells transformed by bovine adenovirus type 3 and its oncogenic DNA fragments, and their clonal normal counterparts, were tested for tumor angiogenesis activity. The activity was assayed by measuring the host-mediated vascular response of a chorioallantoic membrane to the cell suspension separated from the vascular bed by a Millipore filter. Angiogenesis activity due to inflammation reaction was prevented by using corticosteroids. All of the transformed cells tested induced strong vascular responses as compared with their corresponding clonal normal cells. Cell dose and time dependency for the expression of the activity were also shown.

Dependence on chain length of antitumor activity of (1 leads to 3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes, IFO 13140, and its acid-degraded products.

The well-defined (1 leads to 3)-beta-D-glucan with (DPn) 540, produced by cultivation of Alcaligenes faecalis var. Myxogenes (IFO 13140), a mutant of a soil bacterium, had marked inhibitory activity against the s.c.-implanted Sarcoma 180 at 5 to 50 mg/kg for 10 days. It also exhibited very high activity in doses of 60 and 100 mg/kg i.p. by a single injection at 7 days after the initial s.c. transplantation of Sarcoma 180. The inhibition ratios observed with Ehrlich carcinoma, NTF (Nakahara-Tokuzen-Fukuoka) reticulum cell sarcoma, and CCM adenocarcinoma were somewhat less but were still significant. On the other hand the treatment failed to inhibit the growth of ascites Sarcoma 180 or to induce prolongation of life span. The mechanism of action of this glucan was considered to be host mediated because of a lack of effect in vitro and also because of the effectiveness of pretreatment of animals by injection before transplantation of a tumor. The results from the bioassay study of the low-molecular-weight (1 leads to 3)-beta-D-glucans prepared from the glucan with number-average degrees of polymerization (DPn) 540 by hydrolysis with formic acid or sulfuric acid showed that the glucans with DPn greater than 50 inhibited the growth of Sarcoma 180 implanted s.c. in mice, whereas the result of the schedule-dependent effect was obtained in the pretreatment of animals by the glucan with DPn 50. By i.v. administration, even the glucan with DPn 16 showed a strong antitumor effect comparable to that of the glucan with DPn 540.

Mechanism of action of 2,2'-(methylenediimino)bis-1,3,4-thiadiazole (NSC 143019), an antitumor agent.

The mechanism of action of 2,2'-(methylenediimino)bis-1,3,4-thiadiazole (NSC 143019) was clarified by studies on its effects on monolayer cultures of growing cells of the mouse cell line BALB/3T3. At concentrations below 50 muM, NSC 143019 specifically inhibited DNA and RNA syntheses without appreciably affecting protein synthesis. The syntheses of DNA and RNA were inhibited equally and concomitantly by the compound. The inhibition was reversed by removal of the compound and was prevented competitively by an equimolar amount of nicotinamide. It was also reversed completely by guanosine (0.1 mM) or deoxyguanosine (0.1 mM) and was reversed partially by xanthosine (1 mM). Other nucleosides did not influence the inhibition. The inhibition of DNA synthesis by NSC 143019 was not due to inhibition of RNA synthesis, and vice-versa. NSC 143019 inhibited the conversion of [8-14C]hypoxanthine to acid-soluble and -insoluble guanine nucleotides but not to adenine nucleotides. It was strongly suggested from these results that at concentrations of NSC 143019 below 50 muM the primary action of this compound might be due to the inhibition of GMP biosynthesis at the step of conversion of IMP to xanthosine 5'-phosphate.

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.

The effect of the crp genotypes on the transformation efficiency in Escherichia coli.

We have found that a significant difference exists in transformation efficiency between the crp+/crp- isogenic pair of strains of Escherichia coli, with the efficiency being much higher in crp- than in crp+. The ratio of transformation efficiency between crp+ and crp- strains depends very little on the plasmid size. This observation suggests that the difference of the transformation efficiency is due to mechanisms other than a crp-regulated endonuclease. The crp gene is one of the first specific genes that have been shown to affect transformation efficiency.

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.

Cold-sensitive phenotype of Escherichia coli cells harboring a plasmid carrying the kil gene of phage lambda brought under control of cI857 gene promoters.

A plasmid (pI-14), containing part of the phage lambda control region (EcoRI-D fragment of lambda cI857) with a large deletion between genes rex and kil, confers a cold-sensitive (cs) phenotype on the host bacteria, whereas the parent plasmid (pMM200) without the deletion made the host bacteria (Escherichia coli strain DOO) high-temperature sensitive. This phenomenon could be explained on the basis of the sequence analysis of the deletion. Upon this deletion, the lambda kil gene, which was originally under the control of the pL promoter, was brought under the control of the lambda cI promoters, resulting in the reversal of the host cell response to temperature. This example shows that gene circuits showing diametrically opposite responses to environmental factors (in this case, temperature) can be constructed from the same elements when the effector gene (here, kil) is connected in different ways to the sensor gene (here, cI857). This cold-dependent killing activity was also dependent on the crp+ state of the host.

A new DNA cloning/sequencing vector with a built-in mechanism for generation of nested deletions using transposon Tn3.

We have constructed a new cloning/sequencing vector suitable for sequencing of DNA fragments too long to be sequenced in a single step. This vector plasmid contains inverted repeats (IR) of transposon Tn3, the kil and cI857 genes of phage lambda, and multiple cloning sites (MCS). Escherichia coli cells harboring plasmids containing nested deletions from one of the Tn3 IR ends, across kil, to variable end points, can be positively selected by plating at 42 degrees C. The deletion products, fractionated according to their size by agarose-gel electrophoresis, can be sequenced by using a synthetic primer whose 3'-end is located within the Tn3 IR, and the total sequence of the insert can be constructed from the partial sequences.

Insertion of IS4 into the tnpA gene of Tn3: sequencing of the insertion site.

Plasmid pMM237 lacks the deletion-forming activity associated with its Tn3 transposase-encoding gene (mpA) [Morita et al., J. Biochem. 101 (1987) 1253-1264]. Analysis of the plasmid DNA showed that pMM237 was generated by insertion of IS4 into the tnpA gene of the original plasmid pMM234. The insertion was accompanied by a direct 12-bp duplication at the target site. The nucleotide sequence around the target site shared only some of the characteristics of previously reported IS4 target sites [Habermann et al., Mol. Gen. Genet. 175 (1979) 369-373; Klaer et al., Mol. Gen. Genet. 181 (1981) 169-175; Georgopoulos et al., Gene 20 (1982) 83-90; Mayaux et al., Gene 30 (1984) 137-146].

The HU protein is not essential for generating deletions adjacent to transposon Tn3.

Escherichia coli mutants deficient in the HU protein were used to test whether this protein is essential for generating DNA deletions adjacent to the ends of Tn3. There were no significant differences in the frequencies of the adjacent DNA deletions in the isogenic series which differ only in the HU loci (hupA or hupB), indicating that the HU protein is not essential for this reaction.

Purification and characterization of the N gene product of bacteriophage lambda.

The N protein (pN) specified by bacteriophage lambda is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from lambda trp transducing phage bearing N- and fed- mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coli protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing the nusA-nusB- mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pN appears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.

A biochemical assay for the transcription-antitermination function of the coliphage lambda N gene product.

An in vitro assay has been developed for the antiterminating activity of the N gene product (pN) of bacteriophage lambda, based on the N-dependent stimulation of trp mRNA synthesis from the DNA templates of lambda trp transducing phages. Using this assay system, we show (a) that the stimulation of trp mRNA synthesis by pN requires the cis-dominant nutL site on the lambda chromosome and (b) that pN can participate in vitro in the formation of functional antiterminating transcription complexes.

Cloning of rat brain protein kinase C complementary DNA.

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Expression of the cDNA encoding lipocortin-like 39 kDa protein of guinea pig neutrophils in yeast. Purification and biological characterization.

The cDNA encoding lipocortin-like 39 kDa protein in guinea pig neutrophils was cloned into a yeast expression vector and the constructed plasmid was introduced into a yeast. The gene was expressed in an eukaryotic cell, yeast Saccharomyces cerevisiae and the recombinant protein was purified and characterized. The purified protein was identical with the native one with respect to the antigenicity and several biochemical properties, such as inhibitory action against phospholipase A2, Ca2+-dependent binding to acidic-phospholipids and F-actin and availability as a substrate for tyrosine kinase (EGF receptor/kinase) and protein kinase C.

Revaluation of endoscopic laser therapy for treatment of early cancer in the stomach.

The efficacy and safety of the endoscopic laser therapy for eradication of early cancer in the stomach was revaluated in fourty-eight patients. In 37 of the 42 patients (88%) treated with the laser therapy alone, the procedure was effective and the complete eradication was confirmed at the end of the follow-up period. In seven of fourteen patients with the incomplete treatment, residual cancer cells were successfully removed by repeated laser therapy. The endoscopic mucosal resection was effective in 21 of the 30 patients (70%) with the early gastric cancer. Interestingly, six of the eleven cases with incomplete treatment with the endoscopic mucosal resection underwent the laser therapy and the complete eradication was achieved in all patients. Although the endoscopic mucosal resection is now the main method for the endoscopic treatment of the early gastric cancer, these data favor offering the endoscopic laser therapy as the combined method with the mucosal resection therapy.

Overproduction and purification of the Tn3 transposase.

The Tn3 transposase accumulated to about 4% of total cell protein in a minicell-producing Escherichia coli strain harboring a transposase overproducer plasmid. This accumulation of the transposase seems to be due to four factors: derepression of transcription resulting from inactivation of the repressor gene (tnpR); efficient translation caused by a mutation within the Shine-Dalgarno (SD) sequence, the dosage effect of the increased plasmid copy number resulting from deletion of the rom gene of the plasmid; and use of a minicell-producing strain as the host. The Tn3 transposase was purified by a procedure involving five steps; sonication, precipitation of protein by adding polyethyleneimine to the sonic supernatant, followed by extraction of transposase fraction with a buffer containing ammonium sulfate, ammonium sulfate precipitation, gel filtration through Sephacryl S-300, and phosphocellulose and DNA-cellulose chromatography. Milligram quantities of pure transposase can be obtained from one gram of wet cells. A small fraction of the accumulated transposase had a blocked amino-terminus and was eluted separately from the normal protein in the chromatography.