scholarly journals Characterization of the Resistance Mechanism and Risk of Fusarium verticillioides to the Myosin Inhibitor Phenamacril

2020 ◽  
Vol 110 (4) ◽  
pp. 790-794 ◽  
Author(s):  
Weichao Ren ◽  
Na Liu ◽  
Yiping Hou ◽  
Baohua Li ◽  
Mingguo Zhou ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusarium Species ◽  
Fusarium Verticillioides ◽  
Single Point ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Binding Mode ◽  
Ear Rot ◽  
Green Fluorescent ◽  
High Level

Fusarium verticillioides is a major pathogen of maize that causes ear rot and produces mycotoxins. Phenamacril is a novel cyanoacrylate fungicide that exhibits favorable activity against Fusarium species. In this study, the phenamacril-resistant mutants of F. verticillioides were obtained by ultraviolet mutagenesis. Single point mutations of S73L or E276K in the myosin-1 FvMyo1 were proven to be responsible for the high-level resistance of F. verticillioides to phenamacril. Phenamacril had a significant impact on the localization of the wild-type FvMyo1 (FvMyo1WT-green fluorescent protein [GFP]), but not on the mutated FvMyo1 (FvMyo1S73L-GFP and FvMyo1E276K-GFP) at the hyphal tips. Molecular docking analysis suggested that mutation (S73L or E276K) in FvMyo1 altered the binding mode and decreased the binding affinity between phenamacril and myosin-1. There was no significant fitness penalty in mycelial growth, conidiation, and virulence of F. verticillioides associated with resistance to phenamacril. The results will enhance our understanding of the resistance mechanism of F. verticillioides to phenamacril and provide new reference data for the management of maize ear rot.

scholarly journals Dynamics and Spatial Distribution of β-Lactamase Expression in Pseudomonas aeruginosa Biofilms

2004 ◽  
Vol 48 (4) ◽  
pp. 1168-1174 ◽  
Author(s):  
Niels Bagge ◽  
Morten Hentzer ◽  
Jens Bo Andersen ◽  
Oana Ciofu ◽  
Michael Givskov ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Resistance Mechanism ◽  
Inducible Promoter ◽  
Monitor System ◽  
Development Of Resistance ◽  
Biofilm Bacteria ◽  
Green Fluorescent ◽  
High Level

ABSTRACT The development of resistance to β-lactam antibiotics is a problem in the treatment of chronic Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. The main resistance mechanism is high-level expression of the chromosomally encoded AmpC β-lactamase of P. aeruginosa cells growing in biofilms. Several genes have been shown to influence the level of ampC expression, but little is known about the regulation of ampC expression in P. aeruginosa biofilms. To study the expression of ampC in P. aeruginosa biofilms, we constructed a reporter that consisted of the fusion of the ampC promoter to gfp(ASV) encoding an unstable version of the green fluorescent protein. In vitro biofilms of P. aeruginosa were exposed to the β-lactam antibiotics imipenem and ceftazidime. Sub-MICs of imipenem significantly induced the monitor system of the biofilm bacteria in the peripheries of the microcolonies, but the centers of the microcolonies remained uninduced. However, the centers of the microcolonies were physiologically active, as shown by experiments with another monitor construction consisting of an arabinose-inducible promoter fused to gfp(ASV). The whole biofilm was induced in the presence of increased imipenem concentrations. Ceftazidime induced the monitor system of the biofilm bacteria as well, but only bacteria in the peripheries of the microcolonies were induced in the presence of even very high concentrations. The experiments illustrate for the first time the dynamic and spatial distributions of β-lactamase induction in P. aeruginosa cells growing in biofilms. Thus, our experiments show that P. aeruginosa cells growing in biofilms constitute a heterogeneous population unit which may create different antibiotic-selective environments for the bacteria in the biofilm.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

High level Purkinje cell specific expression of green fluorescent protein in transgenic mice

2001 ◽  
Vol 115 (6) ◽  
pp. 455-464 ◽  
Author(s):  
Xulun Zhang ◽  
Stephan L. Baader ◽  
Feng Bian ◽  
Wolfgang Müller ◽  
John Oberdick
Keyword(s):  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Purkinje Cell ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Green Fluorescent ◽  
High Level

scholarly journals Genetic diversity of the Neotropical otter (Lontra longicaudis Olfers, 1818) in Southern and Southeastern Brazil

2007 ◽  
Vol 67 (4 suppl) ◽  
pp. 813-818 ◽  
Author(s):  
CS. Trinca ◽  
HF. Waldemarin ◽  
E. Eizirik
Keyword(s):  
Molecular Diversity ◽  
Single Point ◽  
Haplotype Diversity ◽  
Point Mutations ◽  
Conservation Strategies ◽  
Southeastern Brazil ◽  
Nucleotide Variability ◽  
Single Point Mutations ◽  
High Level ◽  
Lontra Longicaudis

The Neotropical otter is one of the least known otter species, and it is considered to be threatened to various degrees throughout its geographic range. Little information exists on the ecological characteristics of this species, and no genetic study has been published about it until now, hampering the design of adequate conservation strategies for its populations. To contribute with genetic information to comprehensive conservation efforts on behalf of L. longicaudis, we characterized the molecular diversity of the 5’ portion of the mtDNA control region in samples from this species collected in Southern and Southeastern Brazil. The sequence analysis revealed a high level of haplotype diversity (h = 0.819; SE = 0.0052) and nucleotide variability ranging from 0.0039 to 0.0067. One of the sampled haplotypes was the most common in both regions and, from this sequence, several other (locally occurring) haplotypes could be derived by single point mutations. No significant genetic differentiation was observed between the Southern and Southeastern regions.

scholarly journals MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis

2020 ◽  
Vol 71 (16) ◽  
pp. 4877-4889
Author(s):  
Jie-Yang Lu ◽  
Shuang-Xi Xiong ◽  
Wenzhe Yin ◽  
Xiao-Dong Teng ◽  
Yue Lou ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Pollen Wall ◽  
Coat Proteins ◽  
Related Family ◽  
Regulatory Cascade ◽  
Pollen Coat ◽  
Green Fluorescent ◽  
High Level ◽  
And Function

Abstract Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen–stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP–oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

scholarly journals In Silico Insights into HIV-1 Vpu-Tetherin Interactions and Its Mutational Counterparts

2019 ◽  
Vol 7 (6) ◽  
pp. 74
Author(s):  
Patil Sneha ◽  
Urmi Shah ◽  
Seetharaman Balaji
Keyword(s):  
Cell Surface ◽  
Binding Affinity ◽  
Hydrophobic Interactions ◽  
Single Point ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Protein Docking ◽  
Host Protein ◽  
Single Point Mutations ◽  
Hiv 1

Tetherin, an interferon-induced host protein encoded by the bone marrow stromal antigen 2 (BST2/CD317/HM1.24) gene, is involved in obstructing the release of many retroviruses and other enveloped viruses by cross-linking the budding virus particles to the cell surface. This activity is antagonized in the case of human immunodeficiency virus (HIV)-1 wherein its accessory protein Viral Protein U (Vpu) interacts with tetherin, causing its downregulation from the cell surface. Vpu and tetherin connect through their transmembrane (TM) domains, culminating into events leading to tetherin degradation by recruitment of β-TrCP2. However, mutations in the TM domains of both proteins are reported to act as a resistance mechanism to Vpu countermeasure impacting tetherin’s sensitivity towards Vpu but retaining its antiviral activity. Our study illustrates the binding aspects of blood-derived, brain-derived, and consensus HIV-1 Vpu with tetherin through protein–protein docking. The analysis of the bound complexes confirms the blood-derived Vpu–tetherin complex to have the best binding affinity as compared to other two. The mutations in tetherin and Vpu are devised computationally and are subjected to protein–protein interactions. The complexes are tested for their binding affinities, residue connections, hydrophobic forces, and, finally, the effect of mutation on their interactions. The single point mutations in tetherin at positions L23Y, L24T, and P40T, and triple mutations at {L22S, F44Y, L37I} and {L23T, L37T, T45I}, while single point mutations in Vpu at positions A19H and W23Y and triplet of mutations at {V10K, A11L, A19T}, {V14T, I18T, I26S}, and {A11T, V14L, A15T} have revealed no polar contacts with minimal hydrophobic interactions between Vpu and tetherin, resulting in reduced binding affinity. Additionally, we have explored the aggregation potential of tetherin and its association with the brain-derived Vpu protein. This work is a possible step toward an understanding of Vpu–tetherin interactions.

scholarly journals Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

2006 ◽  
Vol 72 (9) ◽  
pp. 6088-6093 ◽  
Author(s):  
Helen Rawsthorne ◽  
Kevin N. Turner ◽  
David A. Mills
Keyword(s):  
Fluorescent Protein ◽  
Group Ii Intron ◽  
Multicopy Plasmid ◽  
Bacterial Genomes ◽  
Gfp Expression ◽  
High Frequencies ◽  
Multicopy Integration ◽  
Group Ii ◽  
Green Fluorescent ◽  
High Level

ABSTRACT Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

scholarly journals Iba1+/NG2+ Macrophage-Like Cells Expressing a Variety of Neuroprotective Factors Ameliorate Ischemic Damage of the Brain

2009 ◽  
Vol 30 (3) ◽  
pp. 603-615 ◽  
Author(s):  
Anna Smirkin ◽  
Hiroaki Matsumoto ◽  
Hisaaki Takahashi ◽  
Akihiro Inoue ◽  
Masahiko Tagawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fluorescent Protein ◽  
Artery Occlusion ◽  
Brain Lesions ◽  
Ischemic Lesion ◽  
Ischemic Brain ◽  
Green Fluorescent ◽  
High Level ◽  
Cerebral Artery Occlusion ◽  
The Brain

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1+/NG2+Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.

The FgCYP51B Y123H mutation confers reduced sensitivity to prochloraz and is important for conidiation and ascospore development in Fusarium graminearum

2021 ◽  
Author(s):  
Yanxiang Zhao ◽  
Mengyu Chi ◽  
Huilin Sun ◽  
Hengwei Qian ◽  
Jun Yang ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Binding Mode ◽  
Head Blight ◽  
Important Amino Acid ◽  
Ascospore Development ◽  
Dmi Fungicides ◽  
Blight Disease ◽  
Dmi Resistance

Fusarium graminearum is one of the most important causal agent of Fusarium Head Blight disease and now were controlled mainly by chemicals such as DMI fungicides. FgCYP51B is one of the DMI targets in F. graminearum and Tyrosine123 is an important amino acid in Fusarium graminearum CYP51B, located in one of the predicted substrate binding pockets based on the binding mode between demethylation inhibitors (DMIs) and CYP51B. Previous study suggests that resistance to DMI fungicides is primarily attributed to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI-resistance, the FgCYP51B Y123H mutant was generated and analyzed. Results revealed that Y123H mutation led to reduced conidial sporulation and affected ascospore development and moreover, the mutation conferred reduced sensitivity to prochloraz. The qPCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and further deepen our understanding of the DMI resistance mechanism.

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