A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.     

Induced expression of the Candida albicans multidrug resistance gene CDR1 in response to fluconazole and other antifungals

Squamous cell carcinoma (SCC) antigen (Ag) present and expressed in normal epithelium and epithelial tissues is used primarily as tumor marker of SCC of the uterine cervix. In this review, we considered factors interfering in vitro with the collection of samples and assay procedures, benign and malignant nongynecological diseases which may be the cause of elevated serum levels of SCC Ag. Contamination with skin or saliva strongly influences SCC Ag levels. SCC Ag is elevated in several benign lesions, including pulmonary (tuberculosis, adult respiratory distress syndrome, pulmonary infiltration with eosinophilia, sarcoidosis, bronchogenic cyst) and skin (eczema, pemphigus, erythroderma epidermitis, psoriasis) diseases. Elevations are observed in SCC malignancies of the head and neck, esophagus, skin, lung, urothelium, anal canal and vulva.     

Decreased accumulation or increased isoleucyl-tRNA synthetase activity confers resistance to the cyclic beta-amino acid BAY 10-8888 in Candida albicans and Candida tropicalis

BAY 10-8888, a cyclic beta-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related beta-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several Candida tropicalis strains which showed MICs higher than those of other Candida strains. An analysis of the C. tropicalis strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for C. albicans. However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for C. albicans. To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.     

A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice

To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.     

The importance of CD54 and CD86 costimulation in T cells stimulated with Candida albicans and Dermatophagoides farinae antigens in patients with atopic dermatitis

A number of studies have demonstrated an increased frequency of allergen-specific T cells producing increased amounts of interleukin-4 (IL-4) and IL-5, but little interferon-y in both the peripheral blood and skin lesions of patients with atopic dermatitis (AD). In this study, to further clarify the characteristics of T cells obtained from AD patients, we examined the dependency of the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) from AD patients on costimulatory molecules. The antigens used were Candida albicans and Dermatophagoides farinae, for which AD patients show increased levels of IgE antibodies. PBMC from control healthy donors stimulated with these antigens incorporated [3H]-thymidine much more than PBMC from AD patients. The addition of anti-CD54, -CD40, -CD80 and -CD86 monoclonal antibodies to the cultures showed that the PBMC required only CD54 and CD86 for stimulation with C. albicans, but required CD54, CD80 and CD86 for stimulation with D. farinae. Among these monoclonal antibodies, the anti-CD54 antibody suppressed the proliferative responses of most PBMC, most effectively followed by the anti-CD86 antibody. However, there were no significant differences in the requirement for costimulatory molecules of PBMC proliferation stimulated with C. albicans or D. farinae between AD patients and healthy donors. Since many studies have suggested that T-helper type 1 and T-helper type 2 immune responses are different in their dependency on CD80 or CD86 costimulation, our present results suggest that the allergen-specific T cells of AD patients are not completely shifted to a T-helper type 2 subset.     

A homozygous mutation in the luteinizing hormone receptor causes partial Leydig cell hypoplasia: correlation between receptor activity and phenotype

Leydig cell hypoplasia (LCH) is characterized by a decreased response of the Leydig cells to LH. As a result, patients with this syndrome display aberrant male development ranging from complete pseudohermaphroditism to males with micropenis but with otherwise normal sex characteristics. We have evaluated three brothers with a mild form of LCH. Analysis of their LH receptor (LHR) gene revealed a homozygous missense mutation resulting in a substitution of a lysine residue for a isoleucine residue at position 625 of the receptor. In vitro analysis of this mutant LHR, LHR(I625K), in HEK293 cells indicated that the signaling efficiency was significantly impaired, which explains the partial phenotype. We have compared this mutant LHR to two other mutant LHRs, LHR(A593P) and LHR(S616Y), identified in a complete and partial LCH patient, respectively. Although the ligand-binding affinity for all three mutant receptors was normal, the hormonal response of LHR(A593P) was completely absent and that of LHR(S616Y) and LHR(I625K) was severely impaired. Low cell surface expression explained the reduced response of LHR(S616Y), while for LHR(I625K) this diminished response was due to a combination of low cell surface expression and decreased coupling efficiency. For LHR(A593P), the absence of a reduced response resulted from both poor cell surface expression and a complete deficiency in coupling. Our experiments further show a clear correlation between the severity of the clinical phenotype of patients and overall receptor signal capacity, which is a combination of cell surface expression and coupling efficiency.     

Gibberellin A3 causes a decrease in the accumulation of mRNA for ACC oxidase and in the activity of the enzyme in azuki bean (Vigna angularis) epicotyls

Differential screening, aimed at the isolation of cDNA clones of mRNAs whose accumulation is influenced by GA3, resulted in the isolation of a cDNA clone of an mRNA whose level was decreased by GA3 in segments of epicotyls of Vigna angularis. The putative protein encoded by this cDNA resembled the 1-aminocyclopropane-1-carboxylate oxidases (ACC oxidases) identified in other plant species (about 80% homology at the amino acid level). Thus, the corresponding gene was designated AB-ACO1 (azuki bean ACC oxidase). GA3 also decreased the activity of ACC oxidase in azuki bean epicotyls, but it did not decrease the rate of ethylene evolution. In fact, GA3 increased the rate of ethylene evolution and the level of ACC. Thus, GA3 seemed to increase the production of ethylene by promoting the synthesis of ACC.     

The role of Ly49A and 5E6(Ly49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts

Due to affluence and a sedentary life style a great deal of people in the western countries are affected by coronary heart disease (CHD). The relation between CHD and certain risk factors pertaining to life style is evaluated in this study. A primary purpose is to study certain crucial risk factors for women. The main variables are age, smoking, overweight (measured by BMI), blood pressure and exercise. This prospective study is based on self-reported data from the nation-wide Swedish Level of Living Survey and on data from the national Cause of Death Register. The data were analysed separately by sex using a proportional hazards model. The sample was divided into two strata: those with heart disease and/or diabetes initially, and all the rest. A sample of 2546 men and 2760 women between 45 and 74 years of age was followed from 1980 to the end of 1990. During this period 189 men and 75 women died of coronary heart disease (CHD). It was found that high blood pressure raised the relative risk (RR) of death from CHD by almost 60% in both men and women. Male smokers (> 14 cigarettes a day) had about 60% (significant) and female smokers (> 10 cigarettes a day) 150% (significant) excessive mortality from CHD. Different levels of overweight among women were strongly related to excess mortality from CHD, ranging between 100 and 300%. Among men there was no such relation. Lack of physical activity showed only a weak (non-significant) increased risk of death due to CHD. Diabetes was also found to be an important risk factor for mortality from CHD, especially among women, being seven times as high as among non diabetics. A test of sex differences revealed that there were two significant interactions, namely between sex and overweight, and between sex and age. Background variables in relation to mortality from all cardiovascular diseases (CVD) were also studied. There were of course many similarities between the effects of the background variables in both the disease groups, but there were interesting differences too, e.g. overweight turned out to be a significant risk factor also for men and physical inactivity for women.