Sensitive Wild Type Research Articles

Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal

BackgroundAspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant.ResultsIn this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress.ConclusionsOur data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.

Ki67 as a Predictor of Response to PARP Inhibitors in Platinum Sensitive BRCA Wild Type Ovarian Cancer: The MITO 37 Retrospective Study.

There is compelling need for novel biomarkers to predict response to PARP inhibitors (PARPi) in BRCA wild-type (WT) ovarian cancer (OC). MITO 37 is a multicenter retrospective study aiming at correlating Ki67 expression at diagnosis with a clinical outcome following platinum treatment and PARPi maintenance. Clinical data were collected from high grade serous or endometroid BRCAWT OC treated with niraparib or rucaparib maintenance between 2010-2021 in 15 centers. Ki67 expression was assessed locally by certified pathologists on formalin-fixed paraffin embedded (FFPE) tissues. Median Ki67 was used as a cut-off. A total of 136 patients were eligible and included in the analysis. Median Ki67 was 45.7% (range 1.0-99.9). The best response to platinum according to median Ki67 was 26.5% vs. 39.7% complete response (CR), 69.1% vs. 58.8% partial response (PR), 4.4% vs. 1.5% stable disease (SD). The best response to PARPi according to median Ki67 was 19.1% vs. 36.8% CR, 26.5% vs. 26.5% PR, 26.5 vs. 25% SD, 27.9% vs. 16.2% progressive disease (PD). No statistically significant differences in progression free survival (PFS) and overall survival (OS) were identified between low and high Ki67. PFS and OS are in line with registration trials. Ki67 at diagnosis did not discriminate responders to PARPi.

590P Ki67 as a predictor of response to PARP inhibitors in platinum sensitive BRCA wild type ovarian cancers: MITO 37 retrospective study

590P Ki67 as a predictor of response to PARP inhibitors in platinum sensitive BRCA wild type ovarian cancers: MITO 37 retrospective study

Unloading outer hair cell bundles in vivo does not yield evidence of spontaneous oscillations in the mouse cochlea

Along with outer hair cell (OHC) somatic electromotility as the actuator of cochlear amplification, active hair bundle motility may be a complementary mechanism in the mammalian auditory system. Here, we searched the mouse cochlea for the presence of spontaneous bundle oscillations that have been observed in non-mammalian ears. In those systems, removal of the overlying membrane is necessary for spontaneous bundle oscillations to manifest. Thus, we used a genetic mouse model with a C1509G (cysteine-to-glycine) point mutation in the Tecta gene where the tectorial (TM) is lifted away from the OHC bundles, allowing us to explore whether unloaded bundles spontaneously oscillate. We used VOCTV in vivo to detect OHC length changes due to electromotility as a proxy for the spontaneous opening and closing of the mechanoelectrical transduction (MET) channels associated with bundle oscillation. In wild type mice with the TM attached to OHC bundles, we did find peaks in vibratory magnitude spectra. Such peaks were not observed in the mutants where the TM is detached from the OHC bundles. Statistical analysis of the time signals indicates that these peaks do not signify active oscillations. Rather, they are filtered responses of the sensitive wild type cochlea to weak background noise. We therefore conclude that, to the limits of our system (∼30 pm), there is no spontaneous mechanical activity that manifests as oscillations in OHC electromotility within the mouse cochlea, arguing that unloaded OHC bundles do not oscillate in vivo.This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

X-ray crystal structure analysis of elacestrant (RAD1901), a novel selective estrogen receptor degrader (SERD), bound to estrogen receptor alpha ligand binding domain.

e15647 Background: Antiestrogens are a mainstay of treatment for estrogen receptor positive (ER+) breast cancer in both the adjuvant and the advanced/metastatic settings. Elacestrant is a mixed activity selective estrogen receptor (SER) alpha (ERα) antagonist, acting as a SER modulator (SERM) at low doses and a SER degrader (SERD) at high doses. It has shown activity in hormone sensitive wild type (WT) ERα and insensitive estrogen receptor gene 1 (ESR1) mutation-harboring (Y537S and D538G) ERα breast cancer, both in preclinical models and in clinical studies. It also possesses a unique pharmacology compared to other competitive ER antagonists in its ability to cross the blood brain barrier. Competitive ERα antagonists are typically comprised of a core that sits in the ligand binding pocket and an arm that manipulates the structure to achieve SERM or SERD activities. In these molecules, the arm is attached in the same position as the triphenylethylene core of tamoxifen. However, elacestrant possesses a novel site of attachment. As such, we hypothesized that elacestrant adopts an alternative binding orientation in the ERα ligand binding pocket to achieve its unique pharmaceutical profiles. Methods: X-ray crystallography was used to solve a co-crystal structure of elacestrant in complex with WT ERα ligand binding domain to 2Å. Results: Overall, elacestrant promotes the formation of a canonical ERα ligand binding domain antagonist conformation, whereby helix 12 (H12) is docked into the activating function-2 cleft. However, elacestrant adopts a novel vector in the ERα ligand binding pocket that places it in close proximity to helix 12. As a result, it forms a bifurcated hydrogen bond that is not observed in other competitive antiestrogens and samples a chemical space known to increase H12 mobility and induce SERD activity. This novel vector also places it near positions 537 and 538, the two most common sites of somatic mutation. Conclusions: The high-resolution x-ray crystal structure of elacestrant suggests that the unique binding mode it adopts enables novel pharmacology and positions it to achieve potency in the WT and activating somatic ERα mutated breast cancer setting.

Utilization of GC-MS untargeted metabolomics to assess the delayed response of glufosinate treatment of transgenic herbicide resistant (HR) buffalo grasses (Stenotaphrum secundatum L.).

Herbicide resistant (HR) buffalo grasses were genetically engineered to resist the non-selective herbicide, glufosinate in order to facilitate a modern, 'weeding program' which is highly effective in terms of minimizing costs and labor. The resistant trait was conferred by an insertion of the pat gene to allow for the production of the enzyme phosphinothricin acetyltransferase (PAT) to detoxify the glufosinate inhibitive effect. To date, there are only a few reports using metabolomics as well as molecular characterizations published for glufosinate-resistant crops with no reports on HR turfgrass. Therefore, for the first time, this study examines the metabolome of glufosinate-resistant buffalo grasses which not only will be useful to future growers but also the scientific community. A major aim of this present work is to characterize and evaluate the metabolic alterations which may arise from a genetic transformation of HR buffalo grasses by comprehensively using gas chromatography-mass spectrometry (GC-MS) based untargeted metabolomics. Eight-week old plants of 4 HR buffalo grasses, (93-1A, 93-2B, 93-3C and 93-5A) and 3 wild type varieties (WT 8-4A, WT 9-1B and WT 9-1B) were selected for physiological, molecular and metabolomics experiments. Plants were either sprayed with 1, 5, 10 and 15% v/v of glufosinate to evaluate the visual injuries or submerged in 5% v/v of glufosinate 3days prior to a GC-MS based untargeted metabolomics analysis. In contrast, the control group was treated with distilled water. Leaves were extracted in 1:1 methanol:water and then analysed, using an in-house GC-MS untargeted workflow. Results identified 199 metabolites with only 6 of them (cis-aconitic acid, allantoin, cellobiose, glyceric acid, maltose and octadecanoic acid) found to be statistically significant (p < 0.05) between the HR and wild type buffalo grass varieties compared to the control experiment. Among these metabolites, unusual accumulation of allantoin was prominent and was an unanticipated effect of the pat gene insertion. As expected, glufosinate treatment caused significant metabolic alterations in the sensitive wild type, with the up-regulation of several amino acids (e.g. phenylalanine and isoleucine) which was likely due to glufosinate-induced senescence. The aminoacyl-tRNA biosynthetic pathway was identified as the most significant enriched pathway as a result of glufosinate effects because a number of its intermediates were amino acids. HR buffalo grasses were very similar to its wild type comparator based on a comprehensive GC-MS based untargeted metabolomics and therefore, should guarantee the safe use of these HR buffalo grasses. The current metabolomics analyses not only confirmed the effects of glufosinate to up-regulate free amino acid pools in the sensitive wild type but also several alterations in sugar, sugar phosphate and organic acid metabolism have been reported.

Abstract 765: Multiple signalling pathways are active in BRAF/NRAS wild type melanomas

Abstract In the last five years, the treatment of advanced melanoma has been revolutionised by the introduction of selective kinase inhibitors and immunotherapy. Selective inhibition of the mitogen activated protein kinase (MAPK) pathway has significantly improved the survival of patients with BRAFV600-mutant melanoma, and combination MEK and CDK4 inhibition is currently being trialed in patients with NRAS-mutant melanoma. Almost a quarter of all melanomas, however, are wild type for BRAF and NRAS and have no known actionable mutations. Currently, patients with wild type melanoma are treated with immunotherapies, which produce prolonged responses, but only in a small subset of patients. Overall, the prognosis for patients with wild type melanomas remains poor, and there is an urgent need to establish better therapeutic strategies for these patients. In order to define new therapeutic targets in BRAF/NRAS wild type melanoma, we sought to characterize the key proliferative and survival pathways in wild type melanomas. Wild type (n=14) and BRAFV600 (n=7) melanomas were treated with the MEK inhibitor trametinib at increasing doses from 0.5nM to 5000nM for 72h and sensitivity to MEK inhibition was assessed using viability assays. Signaling pathway activity in response to trametinib (10nM, 24h) was also examined in our panel of melanoma cells using the Human Phospho-Kinase Antibody Array (R&amp;D Systems), which detects 43 different phosphorylated kinases. As expected, BRAFV600 melanomas were exquisitely sensitive to trametinib treatment (IC50=0.8-2.4nM). We also found that although a subset of BRAF/NRAS wild type melanomas (5/14; 36%) were sensitive to trametinib (IC50 0.5-4.9nM), the majority of these melanoma cells (9/14; 64%) were resistant to trametinib (IC50 10.5-264.8nM). Analysis of kinase activation status revealed little difference in signaling pathway activity between the sensitive and resistant wild type melanomas at baseline. Resistant wild type melanomas had higher activity of STAT3 compared to the sensitive cell lines. However, all these wild type melanomas showed variable activation of multiple signaling pathways that were not active in BRAFV600 melanomas. Specifically, at baseline, wild type melanomas showed pronounced activation of the JNK and p38 MAPK pathways, the FAK/Src pathway (i.e. FAK, Lck, Lyn, Fgr and Yes), the epidermal growth factor receptor tyrosine kinase and β-catenin compared to BRAFV600 melanomas. Activation of multiple signaling pathways in wild type melanomas may contribute to innate resistance or limit the efficacy of MEK inhibition, and we are currently exploring the impact of these pathways on the survival and MEK dependence of wild type melanomas. Citation Format: Zizhen Ming, Su Yin Lim, Richard F. Kefford, Helen Rizos. Multiple signalling pathways are active in BRAF/NRAS wild type melanomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 765.

Photoperiod response and floral transition in sorghum

ABSTRACTSorghum is a short day plant with strong photoperiod response and its cultivation for grain in temperate regions necessitated the development of photoperiod insensitive mutants that can flower rapidly in the long days of summer. Wild type genotypes grow vegetatively in summer accumulating significant biomass before floral transition ensues during the shorter days of fall. Thus, photoperiod insensitive mutants are grown for grain production while photoperiod sensitive wild type genotypes are grown for forage and biomass feedstock production in the United States. However, the molecular mechanism of photoperiod response and floral transition is poorly understood in sorghum. We have previously reported 3 FLOWERING LOCUS T homologues (SbFT1, SbFT8 and SbFT10) that serve as the ultimate mediators of photoperiod response and floral transition, but more work remains to be done to clearly define the molecular function of the upstream regulatory factors. One of the major QTL that accounts for 85% of the flowering time variation, which was reported to be encoding the PRR37 protein is now debated to be encoding the SbFT12 protein, raising further questions as to how SbFT12 may regulate sorghum florigens. Further molecular analyses will uncover the true nature of the day length sensors in sorghum and the mechanisms of their interactions with florigens to modulate photoperiod dependent vegetative growth and floral transition.

Computational techniques are valuable tools for the discovery of protein–protein interaction inhibitors: The 14-3-3σ case

Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3σ was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3σ, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3σ/c-Abl protein-protein interaction.

Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis

Antibiotic resistance and antioxidant defense were induced by ciprofloxacin in planktonic Proteus mirabilis and compared with the natural antibiotic resistance of biofilm. Resistant variants (1X and 1Y) were obtained from cultures of the sensitive wild type “wt” strain 1 in the presence of the antibiotic. Planktonic strain 1 exhibited oxidative stress with increases in the reactive oxygen species (ROS) and consumption of NO in the presence of ciprofloxacin, whereas 1X and 1Y suffered non-significant rises in ROS generation, but produced and consumed more NO than sensitive strain 1. The two resistant variants were more resistant to telluride than wt and showed increased levels of intracellular superoxide dismutase (SOD) and glutathione (GSH). However, ciprofloxacin did not stimulate oxidative stress in biofilm. The production of ROS and NO with or without ciprofloxacin was less significant in biofilms than in an equivalent number of planktonic bacteria; sensitive and resistant strains did not present differences. On the other hand, SOD and GSH were more elevated in the biofilm than in planktonic bacteria. In summary, these results indicate that ciprofloxacin can induce resistance by the enhancement of antioxidant defense in planktonic bacteria, similar to the natural resistance occurring in biofilm. This feature may be added to the factors that regulate the susceptibility to this antibiotic.

Identification of differentially expressed genes associated with 5-methyltryptophan resistance in rice mutants

5-Methyltryptophan (5MT), a tryptophan analog, resistant M4 rice mutants with high free amino acid contents were obtained through in vitro mutagenesis. To evaluate the 5MT resistance mechanism, a cDNA library was constructed by using the leaves and roots of the 5MT resistant mutant plants. Expressed sequenced tags (ESTs) of 1 019 randomly selected clones were analyzed and then assembled 588 unigens. A total of 389 unigenes had significant homologies with known protein sequences at the NCBI database and the remaining 199 unigenes were designated unidentified genes. These unigens were grouped into 13 categories according to their putative functions. Of the 233 randomly selected clones, 25 were identified as differentially expressed genes between 5MT resistant and 5MT sensitive wild type plants. For further study of the differential expression of the genes, expression patterns of 12 genes related to various biological functions were evaluated in response to 5MT treatment in both the resistant plants and sensitive plants. All of the tested 12 genes exhibited higher expression levels in mutant plants than wild type plants under the 5MT inhibition. These expression patterns of the 12 genes suggested that the genes related to 5MT resistance in the rice mutants have a variety of functions, and yield remarkably diverse expression patterns upon 5MT treatment. Many genes that were identified tend to be related to defense and stress responses, suggesting "cross-talking" between biotic/abiotic stresses including the 5MT treatment. Therefore, 5MT resistant mutants might be of value for identifying genes related to plant defenses and stresses.

HIV-2 Protease resistance defined in yeast cells

BackgroundInhibitors of the HIV-1 Protease currently used in therapeutic protocols, have been found to inhibit, although at higher concentrations, the HIV-2 encoded enzyme homologue. Similar to observations in HIV-1 infected individuals, therapeutic failure has also been observed for some patients infected with HIV-2 as a consequence of the emergence of viral strains resistant to the anti-retroviral molecules. In order to be able to define the specific mutations in the Protease that confer loss of susceptibility to Protease Inhibitors, we set up an experimental model system based in the expression of the viral protein in yeast.Results Our results show that the HIV-2 Protease activity kills the yeast cell, and this process can be abolished by inhibiting the viral enzyme activity. Since this inhibition is dose dependent, IC50 values can be assessed for each anti-retroviral molecule tested. We then defined the susceptibility of HIV-2 Proteases to Protease Inhibitors by comparing the IC50 values of Proteases from 7 infected individuals to those of a sensitive wild type laboratory adapted strain.Conclusion This functional assay allowed us to show for the first time that the L90M substitution, present in a primary HIV-2 isolate, modifies the HIV-2 Protease susceptibility to Saquinavir but not Lopinavir. Developing a strategy based on the proposed yeast expressing system will contribute to define amino acid substitutions conferring HIV-2 Protease resistance.

Virulence of antibiotic-resistant Salmonella typhimurium.

We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice. Of seven resistant mutants examined, six were avirulent and one was similar to the wild type in competition experiments in mice. The avirulent-resistant mutants rapidly accumulated various types of compensatory mutations that restored virulence without concomitant loss of resistance. Such second-site compensatory mutations were more common then reversion to the sensitive wild type. We infer from these results that a reduction in the use of antibiotics might not result in the disappearance of the resistant bacteria already present in human and environmental reservoirs. Thus, second-site compensatory mutations could increase the fitness of resistant bacteria and allow them to persist and compete successfully with sensitive strains even in an antibiotic-free environment.

An outbreak of a penicillin-sensitive strain of gonorrhoea in Sydney men.

To describe an outbreak of gonorrhoea caused by penicillin sensitive Wild type 1B2-Wt/1B2(FS), in homosexual men in Sydney. The study sample comprised all gonococcal isolates referred to the Gonococcal Reference Laboratory (GRL), New South Wales, Australia between 1 January 1990 and 30 June 1992. Demographic data on Wt/1B2(FS) were sought by review of all request forms accompanying specimens to the GRL. Detailed review was undertaken of the clinical records of all men with gonorrhoea which had been differentiated by auxotype and serotype (A/S) from two large STD clinics in Sydney. The first isolation of Wt/1B2(FS) was made in April 1990 and by the end of July 1992, 140 such isolates were identified in 131 patients. The male:female ratio was 130:1 suggesting male homosexual transmission, which was confirmed in 55 of 57 evaluable cases. There was a higher proportion of pharyngeal and anal infections among Wt/1B2(FS) isolates compared with all other male gonorrhoea. Demographic information showed that 60% of men with Wt/1B2(FS) were under the age of 30, and 80% lived in Central or Eastern Sydney. An outbreak of a penicillin sensitive strain of gonorrhoea has occurred in Sydney, primarily among gay men living in the inner city. The extent to which the outbreak represents an increase in the risk of HIV transmission is unclear.

Molecular cloning of a glutathione S-transferase overproduced in an insecticide-resistant strain of the housefly (Musca domestica)

We report the cloning and sequencing of a glutathione S-transferase (GST) gene from the housefly Musca domestica. A cDNA lambda gt11 library was prepared from the organophosphate insecticide-resistant housefly strain Cornell-R--a variant that has elevated GST activity. The lambda phage GST clone was identified on the basis of its ability to cross-hybridize to a GST DNA probe from Drosophila melanogaster. Based on amino acid homology to other GSTs and expression of GST activity in Escherichia coli, the Musca GST gene (MdGST-1) belongs to the GST gene family. Although organophosphate resistance in Cornell-R is largely due to one of the GSTs, MdGST-1 is probably not the enzyme responsible for resistance. The mutation that controls resistance to organophosphate insecticides in Cornell-R is highly unstable and we isolated spontaneous variants to both insecticide sensitivity and to even higher levels of resistance. This provided us with an isogenic set of three strains. We found that MdGST-1 transcript levels as measured by Northern assays are higher in all three Cornell-R strains relative to the sensitive wild type, but that the sensitive Cornell-R strain has more MdGST-1 transcript than does the highly resistant Cornell-R strain. These data as well as Southern analysis of genomic DNA allow us to conclude: (1) there are multiple GST genes in M. domestica; (2) the natural variant Cornell-R overproduces excess transcript from two and probably more of these genes; and (3) the unstable mutation in Cornell-R influences the levels of multiple GSTs.

Primisulfuron herbicide-resistant tobacco plants: mutant selection in vitro by adventitious shoot formation from culturéd leaf discs

A simple procedure has been developed for the rapid and direct selection of herbicide-resistant mutant plants. The procedure uses adventitious shoot formation from suitable explants, such as leaf discs, on a shoot-inducing culture medium containing a toxic herbicide concentration. Resistant green shoots were thus isolated from tobacco ( Nicotiana tabacum L.) leaf explants cultured on medium containing 100 μg 1 −1 primisulfuron, a new sulfonylurea herbicide. Resistant shoots were recovered from both haploid and diploid explants after UV mutagenesis, as well as without mutagenic treatment. Three mutant plants of separate origin were further analyzed biochemically and genetically. Their acetohydroxyacid synthase (AHAS) enzyme activity was less inhibited by sulfonylurea herbicides than that of unselected, sensitive wild type plants. The extent of inhibition of the AHAS enzyme among the three mutants was different for different sulfonylurea and imidazolinone herbicides suggesting different sites were affected by each mutation. Herbicide tolerance was scored for germinating seedling populations and was found to be inherited as a single dominant nuclear gene. Adventitious shoot formation from cultured leaf discs was used to determine the cross tolerance of mutant plants to various herbicidal AHAS inhibitors. The usefulness of this rapid and direct scheme for mutant selection based on adventitious shoot formation or embryogenesis is discussed.

Comparison between tobacco cell lines either resistant or sensitive (wild type) to exogenous choline: intracellular accumulation of free choline

Tobacco cells grown in vitro as suspension cultures slowly accumulated exogenous choline during the log phase of growth independently of their sensitivity or resistance to inhibitory levels of choline chloride. Cells from resistant lines repeatedly subculture in the presence of choline (10 or 25 mM) displayed an ability to limit the intracellular accumulation of choline and the associated variation of osmotic potential. On the contrary, cells from the sensitive line actively absorbed and metabolized exogenous choline when supplied at 10–25 mM. At the stationary phase of growth, the internal concentration of free choline in sensitive cells was markedly increased as well as the osmotic pressure of cell fluids. After a few generations, cell divisions stopped and mortality rapidly increased.

Combined application of low temperature preparation and electron microscopic autoradiography for the localization of systemic fungicides.

The intracellular localization of the sterol-biosynthesis-inhibiting (SBI) fungicide (3H)triadimenol A is investigated in vitro in the fungus Ustilago avenae. For this purpose low temperature preparation techniques (shock freezing, freeze substitution, embedding in Lowicryl HM20) are combined with conventional electron microscopic (EM) autoradiography. In particular the suitability of Lowicryl HM20 embedded specimens for EM autoradiography with regard to the finestructure preservation is shown. For the localization of (3H)triadimenol the filamentous grain development as well as the application of the gold latensification method resulting in the appearance of spherical silver grains is tested. Fungicide sensitive wild type sporidia of U. avenae are compared with fungicide resistant cells of the mutant r8. A quantitative analysis of the autoradiographs of the wild type developed according to the gold latensification process shows a relatively homogeneous distribution of silver grains over the entire cell. On the other hand, the resistant mutant is characterized by an accumulation of silver deposits over the vacuoles as compared with the lower density of grains over the cell walls and cytoplasm. The data are discussed in the context of possible resistance mechanisms against SBI-fungicides.

Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability.

Plasma membrane vesicles were isolated from col- chicine-resistant mutant lines and sensitive wild type and revertant lines of Chinese hamster ovary cells after controlled cell disruptions. The pressures required to disrupt the mutant cells (150 p.s.i.) similarly were less than for the wild type (350 p.s.i.) or revertant (300 p.s.i.) cells indicating an increased fragility of the drug-re- sistant mutants. In all three cases, however, the final plasma membrane preparations consisted of homoge- neous populations of vesicles ranging between 0.1 and 0.7 pm in diameter and were enriched lo-fold in 5’- nucleotidase and (Na+ + K’)-Mti+ATPase. An integral membrane glycoprotein with a molecular weight of approximately 170,000 (“P-glycoprotein”) was present in vesicles from the drug-resistant cells but not the others. The amount of this protein was related to the degree of drug resistance. The protein was purified by the following procedure: removal of peripheral mem- brane proteins with 35 lll~ lithium diiodosalicylate, solubilization in 0.5% N-dodecylsarcosinate, and frac- tionation by lectin-agarose affinity chromatography using Ricinus communis-I which bound the P-glycopro- tein and Lens culinaris and Pisum sativum lectins which bound other membrane glycoproteins. The amount of purified P-glycoprotein thus obtained ac- counted for approximately 3 to 4% of the total plasma membrane protein in one colchicine-resistant mutant line.

Effect of undecanoic acid on germination of microconidia of wild and undecanoic acid resistance mutant of Trichophyton rubrum.

Effects of undecanoic acid (UDA) on germination of microconidia and elongation of germ tubes in UDA sensitive (udas) wild type Trichophyton rubrum and UDA resistant (udar) mutant derived from it, were studied. UDA inhibited conidial germination of udas and udar strains at 30 microgram/ml and 120 microgram/ml respectively which were minimum inhibitory concentrations of UDA for these two strains. When spores from both udas and udar were germinated in presence of subinhibitory concentration of UDA, germ tube growth was short. The elongation of germ tubes of spores pregerminated in absence of UDA was also inhibited by dose of UDA not sufficient to inhitib germination.