Generation of Variability in Chrysodeixis includens Nucleopolyhedrovirus (ChinNPV): The Role of a Single Variant

The mechanisms generating variability in viruses are diverse. Variability allows baculoviruses to evolve with their host and with changes in their environment. We examined the role of one genetic variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) and its contribution to the variability of the virus under laboratory conditions. A mixture of natural isolates (ChinNPV-Mex1) contained two genetic variants that dominated over other variants in individual larvae that consumed high (ChinNPV-K) and low (ChinNPV-E) concentrations of inoculum. Studies on the ChinNPV-K variant indicated that it was capable of generating novel variation in a concentration-dependent manner. In cell culture, cells inoculated with high concentrations of ChinNPV-K produced OBs with the ChinNPV-K REN profile, whereas a high diversity of ChinNPV variants was recovered following plaque purification of low concentrations of ChinNPV-K virion inoculum. Interestingly, the ChinNPV-K variant could not be recovered from plaques derived from low concentration inocula originating from budded virions or occlusion-derived virions of ChinNPV-K. Genome sequencing revealed marked differences between ChinNPV-K and ChinNPV-E, with high variation in the ChinNPV-K genome, mostly due to single nucleotide polymorphisms. We conclude that ChinNPV-K is an unstable genetic variant that is responsible for generating much of the detected variability in the natural ChinNPV isolates used in this study.

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Genetic diversity in Chrysodeixis includens nucleopolyhedrovirus and factors involved in its generation

10.48035/tesis/2454/40598 ◽

2021 ◽

Author(s):

◽

Eduardo Aguirre Sánchez

Keyword(s):

Genetic Diversity ◽

Genetic Variability ◽

Genetic Variants ◽

Great Variability ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Natural Isolates ◽

K Variant ◽

Chrysodeixis Includens ◽

Present Thesis

In the present thesis, the genetic variability of field-collected isolates of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) was examined. These results have revealed the great variability present within natural ChinNPV isolates, confirm the differences between two different genetic variants from these isolates and, more important, appear to identify the ChinNPV-K variant as a generator of variability in these isolates. In conclusion, a great variability has been revealed within ChinNPV natural isolates, with a single variant showing a key role in the generation of this diversity in a concentration-dependent manner.

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Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646319 ◽

1992 ◽

Vol 68 (05) ◽

pp. 570-576 ◽

Cited By ~ 20

Author(s):

Mary A Selak

Keyword(s):

Neutrophil Elastase ◽

Cell Activation ◽

Thromboxane A2 ◽

Creatine Phosphate ◽

Dense Granule ◽

Cathepsin G ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Low Concentrations ◽

High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

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The P-element-induced silencing effect of KP transposons is dose dependent in Drosophila melanogaster

Genome ◽

10.1139/g11-023 ◽

2011 ◽

Vol 54 (9) ◽

pp. 752-762 ◽

Cited By ~ 7

Author(s):

Alireza Sameny ◽

John Locke

Keyword(s):

Drosophila Melanogaster ◽

Critical Role ◽

Mutagenic Effect ◽

P Element ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

P Elements ◽

Single Well ◽

Dose Dependent

Transposable elements are found in the genomes of all eukaryotes and play a critical role in altering gene expression and genome organization. In Drosophila melanogaster, transposable P elements are responsible for the phenomenon of hybrid dysgenesis. KP elements, a deletion-derivative of the complete P element, can suppress this mutagenic effect. KP elements can also silence the expression of certain other P-element-mediated transgenes in a process called P-element-dependent silencing (PDS), which is thought to involve the recruitment of heterochromatin proteins. To explore the mechanism of this silencing, we have mobilized KP elements to create a series of strains that contain single, well-defined KP insertions that show PDS. To understand the quantitative role of KP elements in PDS, these single inserts were combined in a series of crosses to obtain genotypes with zero, one, or two KP elements, from which we could examine the effect of KP gene dose. The extent of PDS in these genotypes was shown to be dose dependent in a logarithmic rather than linear fashion. A logarithmic dose dependency is consistent with the KP products interacting with heterochromatic proteins in a concentration-dependent manner such that two molecules are needed to induce gene silencing.

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Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00251.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. G814-G821 ◽

Cited By ~ 10

Author(s):

Bi-Guang Tuo ◽

Jimmy Y. C. Chow ◽

Kim E. Barrett ◽

Jon I. Isenberg

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Duodenal Mucosa ◽

Bicarbonate Secretion ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ussing Chambers ◽

Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200405018 ◽

2004 ◽

Vol 167 (1) ◽

pp. 75-85 ◽

Cited By ~ 79

Author(s):

Brenton L. Scott ◽

Jeffrey S. Van Komen ◽

Hassan Irshad ◽

Song Liu ◽

Kirilee A. Wilson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bud Neck ◽

Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Sirt5 deficiency causes post-translational protein malonylation and dysregulated cellular metabolism in chondrocytes under obesity conditions

10.1101/2020.11.30.404103 ◽

2020 ◽

Author(s):

Shouan Zhu ◽

Albert Batushansky ◽

Anita Jopkiewicz ◽

Dawid Makosa ◽

Kenneth M. Humphries ◽

...

Keyword(s):

Cellular Metabolism ◽

Tca Cycle ◽

Gas Chromatography Mass Spectrometry ◽

Dependent Manner ◽

Joint Injury ◽

Primary Chondrocytes ◽

Wide Range ◽

Chondrocyte Metabolism ◽

High Concentrations

ABSTRACTObjectiveObesity accelerates the development of osteoarthritis (OA) during aging and is associated with altered chondrocyte cellular metabolism. The objective of this study was to investigate the role of sirtuin 5 (SIRT5) in regulating chondrocyte protein lysine malonylation (MaK) and cellular metabolism under obesity-related conditions.MethodsMaK and SIRT5 were immunostained in knee articular cartilage of obese db/db mice and different aged C57BL6 mice with or without destabilization of the medial meniscus (DMM) surgery to induce OA. Primary chondrocytes were isolated from 7-day-old WT and Sirt5−/− mice and treated with varying concentrations of glucose and insulin to mimic obesity. Sirt5-dependent effects on MaK and metabolism were evaluated by Western blot, Seahorse Respirometry, and gas/chromatography-mass/spectrometry (GC-MS) metabolic profiling.ResultsMaK was significantly increased in cartilage of db/db mice and in chondrocytes treated with high concentrations of glucose and insulin (GluhiInshi). Sirt5 protein was increased in an age-dependent manner following joint injury, and Sirt5 deficient primary chondrocytes had increased MaK, decreased glycolysis rate, and reduced basal mitochondrial respiration. GC-MS identified 41 metabolites. Sirt5 deficiency altered 13 distinct metabolites under basal conditions and 18 metabolites under GluhiInshi treatment. Pathway analysis identified a wide range of Sirt5-dependent altered metabolic pathways that include amino acid metabolism, TCA cycle, and glycolysis.ConclusionThis study provides the first evidence that Sirt5 broadly regulates chondrocyte metabolism. We observed changes in Sirt5 and MaK levels in cartilage with obesity and joint injury, suggesting that the Sirt5-MaK pathway may contribute to altered chondrocyte metabolism that occurs during OA development.

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Effect of Palmitic Acid on Exosome-Mediated Secretion and Invasive Motility in Prostate Cancer Cells

Molecules ◽

10.3390/molecules25122722 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2722

Author(s):

Ivan V. Maly ◽

Wilma A. Hofmann

Keyword(s):

Prostate Cancer ◽

Palmitic Acid ◽

Cancer Cells ◽

Dependent Manner ◽

Prostate Cancer Cells ◽

Concentration Dependent Manner ◽

Fat Consumption ◽

Progression Markers

High fat consumption can enhance metastasis and decrease survival in prostate cancer, but the picture remains incomplete on the epidemiological and cell-biological level, impeding progress toward individualized recommendations in the clinic. Recent work has highlighted the role of exosomes secreted by prostate cancer cells in the progression of the disease, particularly in metastatic invasion, and also the utility of targeting these extracellular vesicles for diagnostics, as carriers of disease progression markers. Here, we investigated the question of a potential impact of the chief nutritional saturated fatty acid on the exosome secretion. Palmitic acid decreased the secretion of exosomes in human prostate cancer cells in vitro in a concentration-dependent manner. At the same time, the content of some prospective metastatic markers in the secreted exosomal fraction was also reduced, as was the ability of the cells to invade across extracellular matrix barriers. While by themselves our in vitro results imply that on the cell level, palmitic acid may be beneficial vis-à-vis the course of the disease, they also suggest that, by virtue of the decreased biomarker secretion, palmitic acid has the potential to cause unjustified deprioritization of treatment in obese and lipidemic men.

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The Role Of Calcium In Platelet Microtubule Assembly- Disassembly

10.1055/s-0038-1652479 ◽

1981 ◽

Author(s):

M Kikuchi ◽

Y Ikeda ◽

M Handa ◽

S Matsuda ◽

H Muraki ◽

...

Keyword(s):

Intracellular Calcium ◽

Dynamic Equilibrium ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Microtubule Assembly ◽

Base Line ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Transient Decrease

Microtubules exist in a dynamic equilibrium between polymerized and depolymerized forms in human platelets, playing a major role to maintain the discoid shape of platelets. It has been previously shown that the interaction of aggregating agents with platelets leads to a rapid but transient disassembly of microtubules. ( Steiner and Ikeda, J.Clin. Invest. 63:443,1979 ) In this paper, the role of calcium in the equilibrium between assembled and disassembled microtubules was investigated. The respective pools of soluble and polymerized tubulin were “frozen” by addition of a glycerol-dimethyl sulfoxide-containing medium to platelet rich plasma, preincubated with 2 µM A23187 for various time intervals. The two pools of tubulin were estimated by measuring the colchicine binding activities of total and polymerized tubulin according to the method of Wilson.Resting platelets were found to contain 56.2 ± 2.7 µg tubulin per 109 platelets, of which 56.7 % was in polymerized form. Addition of A23187 to platelet rich plasma produced a transient decrease in the pool of polymerized tubulin within 30 sec., followed by a return to base-line values within 2 min.. TMB-8, a known intracellular calcium antagonist, abolished this transient decrease in polymerized tubulin induced by A23187 in a concentration dependent manner, while indomethacin or acetylsalycylic acid did not.These findings may indicate the important role of intracellular calcium in microtubule assembly-disassembly.

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Oxidation of ubiquinol by peroxynitrite: implications for protection of mitochondria against nitrosative damage

Biochemical Journal ◽

10.1042/bj3490035 ◽

2000 ◽

Vol 349 (1) ◽

pp. 35-42 ◽

Cited By ~ 26

Author(s):

Francisco SCHÖPFER ◽

Natalia RIOBÓ ◽

María Cecilia CARRERAS ◽

Beatriz ALVAREZ ◽

Rafael RADI ◽

...

Keyword(s):

Radical Scavenging ◽

Redox Status ◽

Experimental Models ◽

Dependent Manner ◽

Mitochondrial Membranes ◽

Concentration Dependent Manner ◽

Major Pathway ◽

Membrane Bound ◽

Spectroscopy Studies

A major pathway of nitric oxide utilization in mitochondria is its conversion to peroxynitrite, a species involved in biomolecule damage via oxidation, hydroxylation and nitration reactions. In the present study the potential role of mitochondrial ubiquinol in protecting against peroxynitrite-mediated damage is examined and the requirements of the mitochondrial redox status that support this function of ubiquinol are established. (1) Absorption and EPR spectroscopy studies revealed that the reactions involved in the ubiquinol/peroxynitrite interaction were first-order in peroxynitrite and zero-order in ubiquinol, in agreement with the rate-limiting formation of a reactive intermediate formed during the isomerization of peroxynitrite to nitrate. Ubiquinol oxidation occurred in one-electron transfer steps as indicated by the formation of ubisemiquinone. (2) Peroxynitrite promoted, in a concentration-dependent manner, the formation of superoxide anion by mitochondrial membranes. (3) Ubiquinol protected against peroxynitrite-mediated nitration of tyrosine residues in albumin and mitochondrial membranes, as suggested by experimental models, entailing either addition of ubiquinol or expansion of the mitochondrial ubiquinol pool caused by selective inhibitors of complexes III and IV. (4) Increase in membrane-bound ubiquinol partially prevented the loss of mitochondrial respiratory function induced by peroxynitrite. These findings are analysed in terms of the redox transitions of ubiquinone linked to both nitrogen-centred radical scavenging and oxygen-centred radical production. It may be concluded that the reaction of mitochondrial ubiquinol with peroxynitrite is part of a complex regulatory mechanism with implications for mitochondrial function and integrity.

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Mucin inhibitsPseudomonas aeruginosabiofilm formation by significantly enhancing twitching motility

Canadian Journal of Microbiology ◽

10.1139/cjm-2013-0570 ◽

2014 ◽

Vol 60 (3) ◽

pp. 155-166 ◽

Cited By ~ 10

Author(s):

Cecily L. Haley ◽

Cassandra Kruczek ◽

Uzma Qaisar ◽

Jane A. Colmer-Hamood ◽

Abdul N. Hamood

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iv Pili ◽

Dependent Manner ◽

Twitching Motility ◽

Strain Pao1 ◽

Concentration Dependent Manner ◽

Dependent Effect ◽

Type Iv ◽

Biofilm Development

In Pseudomonas aeruginosa, type IV pili (TFP)-dependent twitching motility is required for development of surface-attached biofilm (SABF), yet excessive twitching motility is detrimental once SABF is established. In this study, we show that mucin significantly enhanced twitching motility and decreased SABF formation in strain PAO1 and other P. aeruginosa strains in a concentration-dependent manner. Mucin also disrupted partially established SABF. Our analyses revealed that mucin increased the amount of surface pilin and enhanced transcription of the pilin structural gene pilA. Mucin failed to enhance twitching motility in P. aeruginosa mutants defective in genes within the pilin biogenesis operons pilGHI/pilJK-chpA-E. Furthermore, mucin did not enhance twitching motility nor reduce biofilm development by chelating iron. We also examined the role of the virulence factor regulator Vfr in the effect of mucin. In the presence or absence of mucin, PAOΔvfr produced a significantly reduced SABF. However, mucin partially complemented the twitching motility defect of PAOΔvfr. These results suggest that mucin interferes with SABF formation at specific concentrations by enhancing TFP synthesis and twitching motility, that this effect, which is iron-independent, requires functional Vfr, and only part of the Vfr-dependent effect of mucin on SABF development occurs through twitching motility.

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