Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase 1 during different stages of infection

2012 ◽  
Vol 58 (5) ◽  
pp. 605-616 ◽  
Author(s):  
Zafer Dallal Bashi ◽  
S. Roger Rimmer ◽  
George G. Khachatourians ◽  
Dwayne D. Hegedus
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Galacturonic Acid ◽  
Hydrolytic Enzymes ◽  
Calcium Signalling ◽  
Solid Surfaces ◽  
Factors Affecting ◽  
Carbohydrate Starvation ◽  
Genes Encoding ◽  
Camp Levels

Sclerotinia sclerotiorum releases hydrolytic enzymes that sequentially degrade the plant cuticle, middle lamellae, and primary and secondary cell walls. The cuticle was found to be a barrier to S. sclerotiorum infection, as leaves stripped of epicuticular wax were more rapidly colonized. Consequently, the factors affecting the regulation of genes encoding polygalacturonase 1 (SsPG1) and a newly identified cutinase (SsCUTA) were examined. In vitro, SsCutA transcripts were detected within 1 h postinoculation of leaves, and expression was primarily governed by contact of mycelia with solid surfaces. Expression of SsPg1 was moderately induced by contact with solid surfaces including the leaf, and expression was restricted to the expanding margin of the lesion as the infection progressed. SsPg1 expression was induced by carbohydrate starvation but repressed by galacturonic acid. Glucose supported a basal level of SsPg1 expression but accentuated expression when provided to mycelia used to inoculate leaves. These observations were contrary to earlier reports indicating that glucose repressed SsPg1 expression while galacturonic acid induced expression. Pharmacological studies showed that disruption of calcium signalling affected SsCutA and SsPg1 expression and decreased S. sclerotiorum virulence, whereas elevated cAMP levels reduced virulence without affecting gene expression. The mechanisms involved in coordinating the expression of S. sclerotiorum hydrolytic enzymes throughout the various stages of the infection are discussed.

scholarly journals Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts

2020 ◽  
Vol 21 (8) ◽  
pp. 2747 ◽  
Author(s):  
Nicholas Mikolajewicz ◽  
Svetlana V. Komarova
Keyword(s):  
Mechanical Stimulation ◽  
Calcium Signalling ◽  
Free Calcium ◽  
Osteoblastic Cell ◽  
Uridine Diphosphate ◽  
P2 Receptor ◽  
Mineral Density ◽  
Osteoblastic Cell Line ◽  
Camp Levels

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

scholarly journals Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis

2008 ◽  
Vol 190 (11) ◽  
pp. 3824-3834 ◽  
Author(s):  
Bob Kennedy M. Dass ◽  
Ritu Sharma ◽  
Avinash R. Shenoy ◽  
Rohini Mattoo ◽  
Sandhya S. Visweswariah
Keyword(s):  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Mycobacterium Smegmatis ◽  
Functional Characterization ◽  
Acid Stress ◽  
Logarithmic Phase ◽  
Intracellular Camp ◽  
Genes Encoding ◽  
Camp Levels

ABSTRACT Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3′,5′-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ΔMSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the ΔMSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ΔMSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.

scholarly journals Influence of the capping of biogenic silver nanoparticles on their toxicity and mechanism of action towards Sclerotinia sclerotiorum

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Mariana Guilger-Casagrande ◽  
Taís Germano-Costa ◽  
Natália Bilesky-José ◽  
Tatiane Pasquoto-Stigliani ◽  
Lucas Carvalho ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Biological Activity ◽  
Cell Lines ◽  
Sclerotinia Sclerotiorum ◽  
Hydrolytic Enzymes ◽  
Chemical Characterization ◽  
In Vitro Cytotoxicity ◽  
Phytopathogenic Fungus ◽  
Biogenic Silver Nanoparticles

Abstract Background Biogenic nanoparticles possess a capping of biomolecules derived from the organism employed in the synthesis, which contributes to their stability and biological activity. These nanoparticles have been highlighted for the control of phytopathogens, so there is a need to understand their composition, mechanisms of action, and toxicity. This study aimed to investigate the importance of the capping and compare the effects of capped and uncapped biogenic silver nanoparticles synthesized using the filtrate of Trichoderma harzianum against the phytopathogenic fungus Sclerotinia sclerotiorum. Capping removal, investigation of the composition of the capping and physico-chemical characterization of the capped and uncapped nanoparticles were performed. The effects of the nanoparticles on S. sclerotiorum were evaluated in vitro. Cytotoxicity and genotoxicity of the nanoparticles on different cell lines and its effects on nontarget microorganisms were also investigated. Results The capped and uncapped nanoparticles showed spherical morphology, with greater diameter of the uncapped ones. Functional groups of biomolecules, protein bands and the hydrolytic enzymes NAGase, β-1,3-glucanase, chitinase and acid protease from T. harzianum were detected in the capping. The capped nanoparticles showed great inhibitory potential against S. sclerotiorum, while the uncapped nanoparticles were ineffective. There was no difference in cytotoxicity comparing capped and uncapped nanoparticles, however higher genotoxicity of the uncapped nanoparticles was observed towards the cell lines. Regarding the effects on nontarget microorganisms, in the minimal inhibitory concentration assay only the capped nanoparticles inhibited microorganisms of agricultural importance, while in the molecular analysis of the soil microbiota there were major changes in the soils exposed to the uncapped nanoparticles. Conclusions The results suggest that the capping played an important role in controlling nanoparticle size and contributed to the biological activity of the nanoparticles against S. sclerotiorum. This study opens perspectives for investigations concerning the application of these nanoparticles for the control of phytopathogens.

In-Vitro Effects of Ricinus Communis Seed Kernel Extract On Some Antioxidant And Hydrolytic Enzymes In Nymph And Adult Zonocerus Variegatus (Grasshopper)

2019 ◽  
Vol 3 (1) ◽  
pp. 129-137
Author(s):  
Gbadebo E . Adeleke ◽  
Olaniyi T. Adedosu ◽  
Rachael O. Adeyi ◽  
John O. Fatoki
Keyword(s):  
Ricinus Communis ◽  
Hydrolytic Enzymes ◽  
Crude Enzyme ◽  
Economic Losses ◽  
Seed Kernel ◽  
Zonocerus Variegatus ◽  
Sod Activity ◽  
Enzyme Preparations ◽  
In Vitro Effects

Background: Many plants have been identified for their insecticidal properties as alternatives to synthetic ones, which are toxic to untargeted organisms and environment. Ricinus communis (Castor) has been re-ported to exhibit insecticidal properties against insect pests. Zonocerus variegatus (Grasshopper) is a notable pest of several crops, and has been linked with great economic losses to farmers. The present study investigates the in-vitro toxicity of R. communis seed kernel extract (RCSKE) on the activities of selected antioxidant and hydrolytic enzymes in nymph and adult Zonocerus variegatus (Grasshopper), using cypermethrin (CYPER-M) and chlorpyrifos (CPF) as standard conventional pesticides. Methods: Seed kernel of Ricinus communis (Castor) was subjected to acidified aqueous extraction to obtain the extract (RCSKE). Crude enzyme preparations were obtained from nymph and adult Z. variegatus grass-hoppers. The in-vitro effects of different concentrations (15, 30, 45, 60, 75, 90 and 105μg/ml) each of RCSKE, CYPER-M and CPF on the activities of superoxide dismutase (SOD), catalase (CAT), acetylcholinesterase (AChE) and carboxylesterase (CES) in crude enzyme preparations were estimated spectrophotometrically. The level of statistical significance was 0.05. Results: The RCSKE significantly reduced the in-vitro SOD activity (p < 0.05) in nymph Z. variegatus at all the concentrations, whereas both CYPER-M and CPF significantly reduced the activity only at certain concentrations. The CAT activity in the nymph was significantly decreased by RCSKE and CPF at all the concentrations, but CYPER-M decreased it only at certain concentrations. In adult Z. variegatus, SOD activity was not significantly affected (p > 0.05), while CAT activity was significantly increased (p < 0.05) by the three agents at all the concentrations. The AChE and CES activities in the nymph were significantly reduced by RCSKE, CYPER-M and CPF at all the concentrations. The RCSKE and CPF significantly increased the CES activity, while CYPER-M caused a significant decrease in the activity in adult Z. variegatus. Conclusion: The seed kernel extract of Ricinus communis is an effective pesticidal agent and hence, it could be a source of biopesticide alternative with greater potential than cypermethrin and chlorpyrifos. In addition, the antioxidant, acetylcholinesterase and carboxylesterase enzymes in the nymphs of Z. variegatus grasshoppers are more susceptible to the effect of the extract than in the adult grasshoppers.

FACTORS AFFECTING THE REGENERATION OF PEPPER (CAPSICUM ANNUUM L.)

HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1120G-1120
Author(s):  
J. L. Jacobs ◽  
C. T. Stephens
Keyword(s):  
Growth Hormone ◽  
Silver Nitrate ◽  
Callus Formation ◽  
Leaf Explants ◽  
Regeneration System ◽  
Factors Affecting ◽  
Nitrate Concentrations ◽  
Leaf Differentiation ◽  
Whole Plants

Several growth hormone combinations and silver nitrate concentrations were examined for their effect on regeneration of different pepper genotypes. Primary leaf explants from in vitro seedlings were cultured on a revised Murashige and Skoog medium supplemented with auxin, cytokinin and 1.6% glucose. Combinations of different concentrations of indole-3-acetic acid (IAA), 0-5 mg/l, and 6-benzylaminopurine (BAP), 0-5 mg/l, were tested to determine the most effective medium for shoot primordium formation. Experiments with IAA and BAP did not result in a specific growth hormone combination appropriate for regeneration of all genotypes tested. Of the silver nitrate concentrations tested, 10 mg/l resulted in the best shoot and leaf differentiation and reduced callus formation. Differences in organogenic response of individual genotypes were evaluated on a single regeneration medium. Whole plants were regenerated from 11 of 63 genotypes examined. Based on these experiments, a reproducible regeneration system for pepper was developed with a total of 500 plants regenerated to date.

Antifungal Proteins from Plant Latex

2020 ◽  
Vol 21 (5) ◽  
pp. 497-506
Author(s):  
Mayck Silva Barbosa ◽  
Bruna da Silva Souza ◽  
Ana Clara Silva Sales ◽  
Jhoana D’arc Lopes de Sousa ◽  
Francisca Dayane Soares da Silva ◽  
...  
Keyword(s):  
Cell Walls ◽  
Defense Mechanisms ◽  
Hydrolytic Enzymes ◽  
Fungal Species ◽  
Biologically Active ◽  
Protein Classification ◽  
Fungal Cell ◽  
Antifungal Proteins ◽  
Plant Latex

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants’ defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

Associated microflora of medicinal ferns: biotechnological potentials and possible applications

2016 ◽  
Vol 5 (03) ◽  
pp. 4927 ◽  
Author(s):  
Shubhi Srivastava ◽  
Paul A. K.
Keyword(s):  
Secondary Metabolites ◽  
Growth Promotion ◽  
Biological Activities ◽  
Hydrolytic Enzymes ◽  
In Vitro Production ◽  
Wide Range ◽  
Negative Effect ◽  
Bacteria And Fungi

Plant associated microorganisms that colonize the upper and internal tissues of roots, stems, leaves and flowers of healthy plants without causing any visible harmful or negative effect on their host. Diversity of microbes have been extensively studied in a wide variety of vascular plants and shown to promote plant establishment, growth and development and impart resistance against pathogenic infections. Ferns and their associated microbes have also attracted the attention of the scientific communities as sources of novel bioactive secondary metabolites. The ferns and fern alleles, which are well adapted to diverse environmental conditions, produce various secondary metabolites such as flavonoids, steroids, alkaloids, phenols, triterpenoid compounds, variety of amino acids and fatty acids along with some unique metabolites as adaptive features and are traditionally used for human health and medicine. In this review attention has been focused to prepare a comprehensive account of ethnomedicinal properties of some common ferns and fern alleles. Association of bacteria and fungi in the rhizosphere, phyllosphere and endosphere of these medicinally important ferns and their interaction with the host plant has been emphasized keeping in view their possible biotechnological potentials and applications. The processes of host-microbe interaction leading to establishment and colonization of endophytes are less-well characterized in comparison to rhizospheric and phyllospheric microflora. However, the endophytes are possessing same characteristics as rhizospheric and phyllospheric to stimulate the in vivo synthesis as well as in vitro production of secondary metabolites with a wide range of biological activities such as plant growth promotion by production of phytohormones, siderophores, fixation of nitrogen, and phosphate solubilization. Synthesis of pharmaceutically important products such as anticancer compounds, antioxidants, antimicrobials, antiviral substances and hydrolytic enzymes could be some of the promising areas of research and commercial exploitation.

scholarly journals Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

2021 ◽  
Vol 22 (11) ◽  
pp. 5968
Author(s):  
Egor A. Turovsky ◽  
Maria V. Turovskaya ◽  
Evgeniya I. Fedotova ◽  
Alexey A. Babaev ◽  
Viktor S. Tarabykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nmda Receptor ◽  
Cortical Neurons ◽  
Target Genes ◽  
Cortical Cell ◽  
Inhibitory System ◽  
Selective Agonist ◽  
Genes Encoding ◽  
Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

scholarly journals Silver Nanoparticles and Their Antibacterial Applications

2021 ◽  
Vol 22 (13) ◽  
pp. 7202
Author(s):  
Tamara Bruna ◽  
Francisca Maldonado-Bravo ◽  
Paul Jara ◽  
Nelson Caro
Keyword(s):  
Silver Nanoparticles ◽  
Antibacterial Agents ◽  
Multidrug Resistant ◽  
Secondary Effects ◽  
Cytotoxic Effects ◽  
Factors Affecting ◽  
Gram Positive Bacteria ◽  
Resistant Strains

Silver nanoparticles (AgNPs) have been imposed as an excellent antimicrobial agent being able to combat bacteria in vitro and in vivo causing infections. The antibacterial capacity of AgNPs covers Gram-negative and Gram-positive bacteria, including multidrug resistant strains. AgNPs exhibit multiple and simultaneous mechanisms of action and in combination with antibacterial agents as organic compounds or antibiotics it has shown synergistic effect against pathogens bacteria such as Escherichia coli and Staphylococcus aureus. The characteristics of silver nanoparticles make them suitable for their application in medical and healthcare products where they may treat infections or prevent them efficiently. With the urgent need for new efficient antibacterial agents, this review aims to establish factors affecting antibacterial and cytotoxic effects of silver nanoparticles, as well as to expose the advantages of using AgNPs as new antibacterial agents in combination with antibiotic, which will reduce the dosage needed and prevent secondary effects associated to both.

scholarly journals In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Amber R Paulson ◽  
Maureen O’Callaghan ◽  
Xue-Xian Zhang ◽  
Paul B Rainey ◽  
Mark R H Hurst
Keyword(s):  
Galleria Mellonella ◽  
Functional Enrichment ◽  
Natural Environments ◽  
Insecticidal Toxin ◽  
Heat Stable ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

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