Distribution of therapeutic response in asthma control between oral montelukast and inhaled beclomethasone.

The distribution of responses in study populations provides a novel method of comparing the benefit of two treatments. This 6-week, randomised, placebo-controlled, double-blind study compared the effectiveness of oral montelukast with inhaled beclomethasone in chronic asthma by assessing the distribution and overlap of patient responses to therapy, as measured by a clinical outcome (asthma control days). A total of 730 adult patients with asthma, age 15-65 yrs, with a forced expiratory volume in one second (FEV1) at baseline of 50-85% of predicted and > or = 15% improvement in FEV1 after inhaled beta-agonist were enrolled. After a 2-week placebo run-in period, patients were randomly allocated to receive montelukast (10 mg once daily), inhaled beclomethasone (200 microg twice daily) or placebo. The primary end-point (per cent of asthma control days) was compared between treatments as the overlap in the response distributions. The overlap of the distribution of responses between the montelukast and beclomethasone groups was 89% for per cent asthma control days and 96% for change from baseline in FEV1. The mean (+/-SD) per cent asthma control days in the montelukast and beclomethasone groups was significantly higher than that in the placebo group (placebo 40.0+/-35.8, montelukast 50.7+/-37.1, beclomethasone 57.9+/-36.1). The mean differences between montelukast and placebo, beclomethasone and placebo, and montelukast and beclomethasone were significant. The mean per cent change (+/-SD) from baseline in FEV1 was 12.1+/-18.7 and 13.9+/-20.8 in the montelukast and beclomethasone groups, respectively, and significantly greater than that in the placebo group (6.4+/-20.1); there was no significant difference between the montelukast and beclomethasone groups in mean values or response distribution. There was also no difference among treatment groups in the frequency of adverse experiences. A comparison of the response distribution is an important approach to comparing therapies; montelukast and beclomethasone provided similar response distributions for the end-point of per cent asthma control days over a 6-week treatment period.

Gene- and tissue-specificity of mutation in Big Blue rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene.

In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes. In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat. All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001). Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04). Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations. All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF. The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes. The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver. Environ. Mol. Mutagen. 37:203-214, 2001. Published 2001 Wiley-Liss, Inc.

Evaluation of the transgenic p53+/- mouse for detecting genotoxic liver carcinogens in a short-term bioassay.

The transgenic p53-deficient heterozygous (p53+/-) mouse is prone to both spontaneous and induced tumors and has been proposed for use in a sensitive, short-term (6 months) assay for identifying genotoxic, multispecies carcinogens. It is not clear, however, if a short-term assay with p53+/- mice detects agents that target certain organs, in particular, the liver. In this study, we treated neonatal male p53+/- and p53+/+ mice with the genotoxic carcinogens dimethylnitrosamine (DMN), 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and 6-nitrochrysene (6-NC). In keeping with the methodology of the proposed short-term assay, the p53+/- mice were evaluated for tumors 7 months after treatment. Wild-type neonatal mice treated with genotoxic carcinogens are known to develop tumors within 1 year; hence, the p53+/+ animals used as controls were subjected to pathological examination at 1 year of age. Our results showed that PhIP was not tumorigenic in either group of mice. Liver tumor incidence increased significantly in the p53+/+ mice treated with DMN and 6-NC, indicating that the conditions of the bioassay were conducive to the promotion of liver tumorigenesis. On the other hand, these two chemicals failed to induce a significant increase in liver tumors in the p53+/- mice by seven months. This result suggests that a deficiency in the amount of p53 protein does not lead to accelerated liver tumorigenesis in mice, and contrasts with previous reports that show a decreased latency of tumors in non-liver targets.

Sensitivity of the phiX174 am3 allele in relation to the endogenous Hprt gene for detecting mutation in transgenic mice.

Transgenic mice have been developed containing multiple, chromosomally integrated copies of the phiX174 am3 allele that serve as reporters for in vivo mutation at a single A:T basepair. In this study, we examined the relative sensitivity of the am3 transgene for detecting the in vivo mutagenicity of N-ethyl-N-nitrosourea (ENU). Three-week-old male phiX174 mice were treated with 0, 40, and 160 mg/kg of ENU. After 1, 3, 6, and 9 weeks, animals were killed, their spleens removed, and isolated splenocytes were used to measure mutant frequencies (MFs) in both the am3 allele and the endogenous Hprt gene. For animals treated with 40 mg/kg of ENU, the Hprt assay detected an average 22-fold increase over background, while the am3 MFs averaged threefold above background. With the 160 mg/kg dose, the Hprt assay detected a 54-fold average increase, while a sixfold average increase above background was found for the transgenic locus. We conclude that the sensitivity of the am3 assay to ENU was compromised by the presence of ex vivo mutations. Adjustment of am3 MFs to exclude these ex vivo mutants could enhance the sensitivity of the assay.

Mutational response at the splenic T-lymphocyte hprt locus in mice treated as neonates: contrasting effects of the carcinogens N-ethyl-N-nitrosourea, dimethylnitrosamine, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The newborn mouse tumorigenicity assay, which involves the treatment of animals during the first two weeks after birth and monitoring tumor induction after a year, has been suggested as a cost- and time-effective alternative to the conventional two-year rodent bioassay. In order to evaluate whether or not lymphocyte hprt mutant induction is an accurate predictor of carcinogenicity in the assay, we determined the frequencies of 6-thioguanine-resistant (TGr) lymphocytes in the spleens of mice neonatally treated with the carcinogenic mutagens N-ethyl-N-nitrosourea (ENU), dimethylnitrosamine (DMN), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Male C57BL/6 pups were injected on postnatal days 8 and 15, and the frequency of TGr T-lymphocytes was measured in groups of three animals, sacrificed periodically up to 31 weeks post-treatment. Compared to background frequencies of 1.1-2.9 x 10(-6), mutant frequencies (MFS) reached 155.1 x 10(-6) following a cumulative dose of 49 mg ENU/kg body weight and 172.3 x 10(-6) following a cumulative dose of 142 mg ENU/kg. These results show that TGr lymphocyte mutations can be induced and measured in mice treated as neonates and that the induced MFs found for mice treated neonatally with ENU are comparable with frequencies reported for the treatment of adult animals with the same chemical. In contrast, treatment with the promutagenic and procarcinogenic compounds DMN (at a maximum concentration of 10.5 mg/kg) and PhIP (26.2 mg/kg) did not result in an increase in lymphocyte MF, suggesting that reactive metabolites of these compounds may not be reaching cells that are sensitive for mutation fixation. The results indicate that the lymphocyte hprt assay may fail to predict the carcinogenicity of some test chemicals in the neonatal mouse bioassay.

Susceptibility of C57BL/6 mice to tumorigenicity induced by dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine in the neonatal bioassay.

Male C57BL/6 neonates were treated on days 8 and 15 with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, 6.5 or 26.2 mg/kg) or dimethylnitrosamine (DMN, 2.6 or 10.5 mg/kg). No tumors were seen in PhIP-treated animals at 15 months of age. Liver and lung tumor incidences in DMN-treated animals were 67-79 and 0-7%, respectively. In comparison with data from other strains, our results indicate that (1) neonatally-treated C57BL/6 mice are resistant to the induction of liver and lung tumors by PhIP and lung tumors by DMN and (2) the susceptibility of this strain to induced liver tumors correlates with the activity of hepatic DMN N-demethylase and PhIP N-hydroxylase in the (untreated) neonates.

The mutagenic response at the ouabain resistance locus in T cells of mice exposed to N-ethyl-N-nitrosourea parallels the response at the Hprt locus and correlates with mutation target size.

The lymphocyte Hprt gene has been used extensively as a reporter locus to monitor the mutational effects of the exposure of animals to genotoxicants. Implicit in this view of the function of a reporter gene is the assumption that its mutagenic response is representative of that of other genes in the organism. As a test of this hypothesis we compared the frequency of 6-thioguanine-resistant (TGr) mutants at the Hprt locus with the mutant frequency (MF) induced at another locus, the ouabain resistance (Oua) locus. The frequency of spontaneous OUA(R) mutants was estimated to be 1.1x10(-7) (MF between <0.3 and 1.1x10(-7)), which was approximately 30-fold less than the spontaneous TGr MF. Following treatment with N-ethyl-N-nitrosourea (ENU), the induced OUA(R) MF at each of two dose levels (50 and 150 mg/kg ENU) and two time points (3 and 6 weeks post-exposure) was consistently 8- to 9-fold lower than the corresponding TGr MF. Thus the mutagenic response of the Oua locus closely paralleled that of the Hprt locus, indicating a similarity in their response to ENU. In addition, the Oua locus was 3-4 times more sensitive than the Hprt locus to the mutagenic effect of ENU, as measured by the fold increase in MF over the background level. The number of ENU-mutable sites capable of resulting in a TGr or OUA(R) phenotype, otherwise known as the mutation target size, was estimated to differ by an order of magnitude between the two loci. This difference in target size correlates with, and therefore may largely account for, the difference in induced MF between both loci.

Frequency of spontaneous and induced micronuclei in the peripheral blood of aging mice.

The mouse peripheral blood micronucleus assay, a measure of DNA damage in erythroblastic cells, was used to determine: (1) the incidence of spontaneously occurring micronucleated reticulocytes (MNRETs) as a function of age, and (2) the induction of micronuclei following treatment of young and old animals with mitomycin C. Male C57BL/6 mice, 92 weeks of age, exhibited a significantly higher frequency of spontaneously occurring peripheral blood MNRETs than mice that were 6 or 10 weeks of age. Mice that were 5-6 weeks or 91-92 weeks old were treated with one dose, or two consecutive doses of mitomycin C; this resulted in dose-related increases in the frequency of MNRETs. Mitomycin C, at a single dose of 1 or 2 mg/kg, induced one-third as many MNRETs in the older animals as compared to the younger animals. When treated with a split dose of mitomycin C (total dose 0.5 to 2 mg/kg), older animals displayed on average two-thirds the mutagenic response of the younger animals. However, analysis of variance performed on these data indicated that the age of the animals did not have a significant effect on their mutagenic response to mitomycin C at any dose level. It appears that aging mice may not be more sensitive to the mutagenic effects of chemically-induced DNA damage than younger mice, suggesting that the higher spontaneous mutation frequency in older mice could be the result of an increased load of accumulated DNA damage.

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions.

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethyl-sulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.

Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Electrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF-1767 and ME-446, has been determined using transverse alternating field electrophoresis. The genomic DNA of BKMF-1767 was resolved into 10 chromosomes ranging in size from 1.8-5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME-446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32 Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF-1767 and to four chromosomes in strain ME-446.

Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent.

The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.

Characterization of extracellular peroxidases produced by acetate-buffered cultures of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Growth of Phanerochaete chrysosporium in a nitrogen-limited medium buffered with sodium acetate, instead of the commonly used 2,2-dimethylsuccinate (DMS), resulted in quantitative and qualitative differences in the production of various extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) involved in lignin degradation. The results indicate that production of LIPs and MNPs can be selectively enhanced by manipulation of culture conditions. Partial N-terminal analyses of the major LIPs and MNPs have made it possible to assign a specific protein to the specific genes and cDNAs that have been reported recently. The LIPs and MNPs differed widely in their ability to decolorize various dyes that are known to be degraded by the lignin degrading enzyme system of P. chrysosporium.

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium.

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.

Nitrogen-deregulated mutants of Phanerochaete chrysosporium--a lignin-degrading basidiomycete.

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (14C-synthetic lignin----14CO2), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the other hand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H2O2 in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Intragenic suppression of the temperature-sensitivity caused by a mutation in a gene controlling transcription (fit) in Escherichia coli.

Starting from a transcription-defective strain harbouring a temperature-sensitive mutation in the fit gene, a rifampicin-resistant, temperature-insensitive derivative has been isolated. Genetic analysis of this derivative demonstrated the presence of a second temperature-sensitive mutation in the same gene. The two mutations mutually suppress each other's phenotype. From cotransduction experiments, the fit gene has been mapped 0.32 min and 0.16 min clockwise from the aroD and pps loci, respectively, at 37.5 min on the linkage map. The mutants harbouring either or both of the fit mutations are defective in RNA synthesis at the non-permissive temperature. The fit gene product is suggested to function as an accessory transcription factor.