scholarly journals Monitoring and Tracking Changes in Sensitivity to Azoxystrobin Fungicide in Alternaria solani in Wisconsin

Plant Disease ◽  
2008 ◽  
Vol 92 (4) ◽  
pp. 555-560 ◽  
Author(s):  
N. Rosenzweig ◽  
G. Olaya ◽  
Z. K. Atallah ◽  
S. Cleere ◽  
C. Stanger ◽  
...  
Keyword(s):  
Management Practices ◽  
Alternaria Solani ◽  
Wild Type ◽  
In Vitro Sensitivity ◽  
Commercial Potato ◽  
Repeated Applications ◽  
Polymerase Chain ◽  
Sensitivity Changes ◽  
Sensitivity Decrease

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

Expression of Ara-C Metabolizing Enzymes Mediates in Vitro Sensitivity to Ara-C in Primary AML Cells From Patients with Denovo AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3481-3481
Author(s):  
Ajay Abraham ◽  
Savitha Varatharajan ◽  
Ashok kumar Jayavelu ◽  
Shaji R Velayudhan ◽  
Rayaz Ahmed ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Candidate Genes ◽  
P Value ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutation Status ◽  
Expression Levels ◽  
In Vitro Sensitivity ◽  
Mrna Expression Levels

Abstract Abstract 3481 Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (ara-C) and daunorubicin. The pre-requisite for the cytotoxic action of pro-drug Ara-C is the enzymatic conversion to its active tri-phosphorylated form ara-CTP. Many drug activating (Deoxycytidine kinase (dCK) and human Equilibrative Nucleoside Transporter 1 (hENT1) and deactivating (Cytidine deaminase (CDA), 5'nucleotidase (NT5C2) genes and ribonucleoside reductase (RRM1), which are involved in transport and biotransformation of cytarabine contribute to the variation in ara-C sensitivity in AML patients. FLT3-ITD and NPM1 mutations act as major poor and good prognostic markers respectively in cytogenetically normal AML. The effect of these mutations in ara-C metabolism remains to be elucidated. The present study aims to determine independent as well as the combined effect of ara-C metabolizing genes mRNA expression on in-vitro ara-C cytotoxicity and the role of FLT3-ITD and NPM mutations on mRNA expression of these genes. Diagnostic bone marrow sample (median blasts 65%; range 21 – 98%) from 98 adult patients with de novo AML (other than AML-M3) were included in this study. mRNA expression levels for each target gene relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. In vitro sensitivity or resistance was classified on the basis of the IC-50 values <6uM and >6uM ara-C respectively. FLT3 ITD and NPM mutation status at diagnosis were determined through PCR followed by Genescan analysis using genomic DNA samples. Type of NPM mutation was identified by sequencing. When ara-C IC-50 values were compared with the mRNA expression levels of these candidate genes, Ara-C sensitive samples (n= 30; IC-50 < 6uM) showed significantly higher mRNA expression of dCK and hENT1 compared to those with Ara-C resistance (n=51) IC50 >6uM (median 314 (61.56 – 1232) vs. 180 (31.87 – 749.2); p = 0.0004 and median 172.1 (44.12 – 657.6) vs. 96.19 (37.49 – 432.4), p= 0.0008 respectively. RRM1 and NT5C2 did not show any association with in vitro Ara-C cytotoxicity, while CDA showed a trend towards association with lower CDA expression in ara-C sensitive samples. Based on these findings we put forward Ara-C resistance index (RI). RI is calculated by the formula RI = ΔCT (dCK X ENT1)/ ΔCT CDA. (Smaller ΔCT value= higher mRNA expression). RI values were significantly higher in resistant (IC50 >6uM) compared to sensitive cells (median: 6.084; range 1.89–11.82) vs. 3.702 (1.89–9.80); p=<0.0001). This association should now be validated in an independent cohort. Effects of NPM and FLT3 mutation status on Ara-C metabolizing genes were then evaluated. No significant association was found between FLT3-ITD status and the mRNA expression of these candidate genes. Interestingly, dCK mRNA levels were significantly higher in samples with NPM mutation (n=39) compared to NPM wild type (n=59); median 272.3 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.01. When analysed separately, patients with NPM type A mutation (n=27) showed significantly higher dCK expression (median 347.4 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.003 compared to those with wild type NPM1. This first report showing an association between expression profiles of ara-C metabolizing genes and NPM mutation should form the basis for evaluating their clinical correlations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals C7-07: Presence of amphiregulin autocrine-loop predicts in vitro sensitivity of EGFR wild type NSCLC and HNSCC cell lines to gefitinib and cetuximab

2007 ◽  
Vol 2 (8) ◽  
pp. S382
Author(s):  
Kimio Yonesaka ◽  
Kreshnik Zejnullahu ◽  
Alison J. Homes ◽  
Bruce E. Johnson ◽  
Pasi A. Janne
Keyword(s):  
Cell Lines ◽  
Wild Type ◽  
Autocrine Loop ◽  
In Vitro Sensitivity ◽  
Hnscc Cell

scholarly journals The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves

Development ◽  
2001 ◽  
Vol 128 (10) ◽  
pp. 1771-1783 ◽  
Author(s):  
E. Semiarti ◽  
Y. Ueno ◽  
H. Tsukaya ◽  
H. Iwakawa ◽  
C. Machida ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Apical Meristem ◽  
Homeobox Genes ◽  
Free Medium ◽  
Wild Type ◽  
Leaf Lamina ◽  
Chain Reaction ◽  
Class 1 ◽  
Polymerase Chain

The asymmetric leaves2 (as2) mutant of Arabidopsis thaliana generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner. Both the delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants. A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves. However, several parallel veins of very similar thickness were evident in such leaves. The complexity of venation patterns in all leaf-like organs of as2 plants was reduced. The malformed veins were visible before the development of asymmetry of the leaf lamina and were maintained in mature as2 leaves. In vitro culture on phytohormone-free medium of leaf sections from as2 mutants and from the asymmetric leaves1 (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells. Analysis by the reverse transcription-polymerase chain reaction showed that transcripts of the KNAT1, KNAT2 and KNAT6 (a recently identified member of the class 1 knox family) genes accumulated in the leaves of both as2 and as1 plants but not of wild type. Transcripts of the STM gene also accumulated in as1 leaves. These findings suggest that, in leaves, the AS2 and AS1 genes repress the expression of these homeobox genes, which are thought to maintain the indeterminate cell state in the shoot apical meristem. Taken together, our results suggest that AS2 and AS1 might be involved in establishment of a prominent midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of class 1 knox homeobox genes in leaves.

scholarly journals Effect of the F129L Mutation in Alternaria solani on Fungicides Affecting Mitochondrial Respiration

Plant Disease ◽  
2005 ◽  
Vol 89 (3) ◽  
pp. 269-278 ◽  
Author(s):  
J. S. Pasche ◽  
L. M. Piche ◽  
N. C. Gudmestad
Keyword(s):  
Disease Control ◽  
Mitochondrial Respiration ◽  
Alternaria Solani ◽  
Complex Iii ◽  
In Vivo Studies ◽  
Cross Sensitivity ◽  
In Vitro Sensitivity ◽  
Wide Range

Isolates of Alternaria solani previously collected from throughout the Midwestern United States and characterized as being azoxystrobin sensitive or reduced sensitive were tested for sensitivity to the Quinone outside inhibitor (QoI) fungicides famoxadone and fenamidone and the carboxamide fungicide boscalid. All three fungicides affect mitochondrial respiration: famoxadone and fenamidone at complex III, and boscalid at complex II. A. solani isolates possessing reducedsensitivity to azoxystrobin also were less sensitive in vitro to famoxadone and fenamidone compared with azoxystrobin-sensitive isolates, but the shift in sensitivity was of lower magnitude, approximately 2- to 3-fold versus approximately 12-fold for azoxystrobin. The in vitro EC50 values, the concentration that effectively reduces germination by 50% relative to the untreated control, for sensitive A. solani isolates were significantly lower for famoxadone and azoxystrobin than for fenamidone and boscalid; whereas, for reduced-sensitive isolates, famoxadone EC50 values were significantly lower than all other fungicides. Isolates of A. solani with reducedsensitivity to azoxystrobin were twofold more sensitive in vitro to boscalid than were azoxystrobin-sensitive wild-type isolates, displaying negative cross-sensitivity. All isolates determined to have reduced-sensitivity to azoxystrobin also were determined to possess the amino acid substitution of phenylalanine with leucine at position 129 (F129L mutation) using real-time polymerase chain reaction. In vivo studies were performed to determine the effects of in vitro sensitivity shifts on early blight disease control provided by each fungicide over a range of concentrations. Reduced-sensitivity to azoxystrobin did not significantly affect disease control provided by famoxadone, regardless of the wide range of in vitro famoxadone EC50 values. Efficacy of fenamidone was affected by some azoxystrobin reduced-sensitive A. solani isolates, but not others. Boscalid controlled azoxystrobin-sensitive and reduced-sensitive isolates with equal effectiveness. These results suggest that the F129L mutation present in A. solani does not convey cross-sensitivity in vivo among all QoI or related fungicides, and that two- to threefold shifts in in vitro sensitivity among A. solani isolates does not appreciably affect disease control.

scholarly journals Parasitic Fitness of Fungicide-Resistant and -Sensitive Isolates of Alternaria solani

Plant Disease ◽  
2018 ◽  
Vol 102 (3) ◽  
pp. 666-673 ◽  
Author(s):  
Mitchell J. Bauske ◽  
Neil C. Gudmestad
Keyword(s):  
Spore Germination ◽  
Point Mutations ◽  
Alternaria Solani ◽  
The United States ◽  
Wild Type ◽  
Parasitic Fitness ◽  
Fitness Parameters ◽  
Multiple Chemical

Resistance to chemistries of the succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicides has developed rapidly in populations of Alternaria solani, the cause of early blight of potato. Reduced sensitivity to the anilinopyrimidine (AP) fungicide pyrimethanil has also been identified recently, determining that resistance to three chemical classes of fungicides is present within the A. solani population. Although no mutations have been characterized to confer resistance to APs, in A. solani five point mutations on three AsSdh genes have been determined to convey resistance to SDHIs, and the substitution of phenylalanine with leucine at position 129 (F129L) in the cytb gene confers resistance to QoIs. The objective of this study was to investigate the parasitic fitness of A. solani isolates with resistance to one or more of these chemical classes. A total of 120 A. solani isolates collected from various geographical locations around the United States were chosen for in vitro assessment, and 60 of these isolates were further evaluated in vivo. Fitness parameters measured were (i) spore germination in vitro, (ii) mycelial expansion in vitro, and (iii) aggressiveness in vivo. No significant differences in spore germination or mycelial expansion (P = 0.44 and 0.51, respectively) were observed among wild-type and fungicide-resistant isolates in vitro. Only A. solani isolates possessing the D123E mutation were shown to be significantly more aggressive in vivo (P < 0.0001) compared with wild-type isolates. These results indicate that fungicide-resistant A. solani isolates have no significant fitness penalties compared with sensitive isolates under the parameters evaluated regardless of the presence or absence of reduced sensitivity to multiple chemical classes. Results of these studies suggest that A. solani isolates with multiple fungicide resistances may compete successfully with wild-type isolates under field conditions.

scholarly journals Why MIC matters? Microbiologists vs. clinicians

2013 ◽  
Vol 4 (1S) ◽  
pp. 39-43
Author(s):  
Corrado Girmenia
Keyword(s):  
Clinical Practice ◽  
Antifungal Agents ◽  
Fungal Species ◽  
Wild Type ◽  
Mechanisms Of Resistance ◽  
In Vitro Sensitivity ◽  
Sensitivity Tests ◽  
Type Population ◽  
European Standards

Several mechanisms of resistance may interfere with the patients’ healing from a fungal infection. However, in premature children fungi are generally susceptible to antifungal agents, except in case of vertical transmission from the mother. In vitro sensitivity tests have been recently harmonized between American and European standards, after years of unhomogeneity: to date, the interpretation of epidemiological cut-off of wild-type population and clinical cut-off are common, and are often updated. Sensitivity tests are difficult to perform, but luckily new commercial tests approvedby FDA are available. Sometimes, knowing the intrinsic sensitivities and resistances of every fungal species and subspecies may be enough in the clinical practice, since few resistances are acquired.

scholarly journals In vitro sensitivity of Alternaria solani to conventional fungicides and a biofungicide based on tea tree essential oil

2015 ◽  
Vol 30 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Milos Stepanovic ◽  
Stojan Jevremovic ◽  
Emil Rekanovic ◽  
Milica Mihajlovic ◽  
Svetlana Milijasevic-Marcic ◽  
...  
Keyword(s):  
Essential Oil ◽  
Alternaria Solani ◽  
Tea Tree Oil ◽  
Tea Tree ◽  
In Vitro Sensitivity ◽  
Infected Tomato

A study of in vitro sensitivity of five Alternaria solani isolates to cooper-oxychloride, chlorothalonil, difenoconazole, pyraclostrobin and a biofungicide based on tea tree essential oil was carried out. The isolates were obtained from infected tomato leaves collected from five different locations in Serbia. The tested isolates showed the highest sensitivity to pyraclostrobin with EC50 values ranging from 0.0014 to 0.0041 ?g ml-1. The EC50 values of difenoconazole were 0.018-0.037 ?g ml-1, chlorothalonil 2.99-4.54 ?g ml-1, and cooper-oxychloride 13.27-15.63 ?g ml-1. All tested A. solani isolates were the least sensitive to tea tree oil (1323.97-3307.08 ?g l-1).

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

Triploidy in a Live-Born Extremely Low Birth Weight Twin: Clinical Aspects

2020 ◽  
Author(s):  
Liliya Vakrilova ◽  
Stanislava Hitrova-Nikolova ◽  
Irena Bradinova
Keyword(s):  
Left Lung ◽  
Extremely Low Birth Weight ◽  
Clinical Aspects ◽  
Severe Respiratory Distress ◽  
Vitro Fertilization ◽  
Gestational Week ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction ◽  
Second Twin

AbstractTriploidy is a rare chromosomal aberration characterized by a karyotype with 69 chromosomes. Triploid fetuses usually are miscarried in early pregnancy. We present a case of a triploid twin and a genetically unaffected co-twin, conceived through in vitro fertilization. A discordant growth was registered at 20 weeks of gestation. Cesarean section was performed at 355/7 gestational week. The second twin was extremely growth restricted female (780 g) with oligohydramnios and severe respiratory distress, and died at 20 hours of age. The autopsy revealed unilobar left lung, bilobar right lung, and cysts of the terminal bronchioles. Quantitative fluorescent polymerase chain reaction detected triploidy compatible pattern. So, early intrauterine growth restriction may be a sign of triploidy, which must be proven by pre or postnatal genetic testing.

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