Optimisation of an in vitro antifungal protein assay for the screening of potential antifungal proteins against Leptosphaeria maculans

2011 ◽  
Vol 84 (1) ◽  
pp. 121-127 ◽  
Author(s):  
Vinochani Pillay ◽  
Gideon M. Polya ◽  
German C. Spangenberg
Keyword(s):  
Leptosphaeria Maculans ◽  
Antifungal Protein ◽  
Protein Assay ◽  
Antifungal Proteins

scholarly journals A Novel Secreted Cysteine-Rich Anionic (Sca) Protein from the Citrus Postharvest Pathogen Penicillium digitatum Enhances Virulence and Modulates the Activity of the Antifungal Protein B (AfpB)

2020 ◽  
Vol 6 (4) ◽  
pp. 203
Author(s):  
Sandra Garrigues ◽  
Jose F. Marcos ◽  
Paloma Manzanares ◽  
Mónica Gandía
Keyword(s):  
Citrus Fruit ◽  
Penicillium Digitatum ◽  
Antifungal Protein ◽  
Penicillium Expansum ◽  
Wild Type ◽  
Phenotypic Differences ◽  
Antifungal Proteins ◽  
Ascomycete Fungi ◽  
In Vitro Antifungal Activity

Antifungal proteins (AFPs) from ascomycete fungi could help the development of antimycotics. However, little is known about their biological role or functional interactions with other fungal biomolecules. We previously reported that AfpB from the postharvest pathogen Penicillium digitatum cannot be detected in the parental fungus yet is abundantly produced biotechnologically. While aiming to detect AfpB, we identified a conserved and novel small Secreted Cysteine-rich Anionic (Sca) protein, encoded by the gene PDIG_23520 from P. digitatum CECT 20796. The sca gene is expressed during culture and early during citrus fruit infection. Both null mutant (Δsca) and Sca overproducer (Scaop) strains show no phenotypic differences from the wild type. Sca is not antimicrobial but potentiates P. digitatum growth when added in high amounts and enhances the in vitro antifungal activity of AfpB. The Scaop strain shows increased incidence of infection in citrus fruit, similar to the addition of purified Sca to the wild-type inoculum. Sca compensates and overcomes the protective effect of AfpB and the antifungal protein PeAfpA from the apple pathogen Penicillium expansum in fruit inoculations. Our study shows that Sca is a novel protein that enhances the growth and virulence of its parental fungus and modulates the activity of AFPs.

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.

Prediction of Human Acute Toxicity by the Hep G2/24-hour/Total Protein Assay, with Protein Measurement by the CBQCA Method

2005 ◽  
Vol 33 (3) ◽  
pp. 207-213 ◽  
Author(s):  
Paul J. Dierickx
Keyword(s):  
Protein Content ◽  
Total Protein ◽  
Total Protein Content ◽  
Human Toxicity ◽  
Hep G2 ◽  
Vitro Assay ◽  
Protein Assay ◽  
Lowry Method ◽  
Total Protein Assay

In our previously described Hep G2/24-hour/total protein assay, protein levels were measured by using the Lowry method. This assay was the best acute in vitro assay for the prediction of human toxicity within the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) study. In order to increase the MEIC data-base with a wider range of chemicals, we were interested in introducing the more practical 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA) method for the quantification of the total protein content. Therefore, we investigated whether the same good results for the prediction of acute human toxicity would be obtained with the CBQCA method. The cells were treated for 24 hours, then cytotoxicity was determined by measuring the total protein content with CBQCA. The results were quantified by using the PI50c: the concentration (in mM) of test compound required to reduce the total protein content measured with the CBQCA-method by 50% as compared to the control cells. The results were compared with the PI50, the corresponding value when the Lowry method was used. A relatively low correlation was observed between PI50 and PI50c, reflecting the large and unexpected, differences when using the two protein assays. However, when comparing the log PI50c with the human toxicity, a correlation coefficient of r2 = 0.761 ( n = 44) was obtained for exactly the same series of MEIC chemicals. This value is clearly higher than that for the Lowry method ( r2 = 0.695). Compared to the Lowry method originally used, the Hep G2/24-hour/CBQCA total protein assay has the additional important advantage that it can be very easily adapted for large-scale analyses with robotic systems, including the on-line calculation of the results.

scholarly journals TITANIUM NANOPARTICLES CONJUGATED WITH STREPTOKINASE AS A MODIFIED THROMBOLYTIC AGENT

2020 ◽  
pp. 18-25
Author(s):  
HAYDER H. ABED ◽  
ESTABRAQ AR. ALWASITI ◽  
AMIR T. TAWFEEQ
Keyword(s):  
Thrombolytic Agent ◽  
Blood Clot ◽  
Control Group ◽  
Thrombolytic Activity ◽  
Protein Assay ◽  
Blood Clots ◽  
Ft Ir ◽  
Titanium Nanoparticles ◽  
D Dimer

Objective: Blood clots are the main cause of death worldwide by stroke and myocardial infarction. Streptokinase a thrombolytic agent that is used in the treatment of circulatory disorders. Methods: Titanium Nanoparticles was supplied from Changsha Santech Co. Its characterized were studied using (FT-IR, XRD, AFM, FE-SEM). Streptokinase at concentration 0.1 mg/ml was conjugated with Titanium nanoparticles using PH equal to 5.2 with continuous stirring. Formation of Streptokinase loading Titanium nanoparticles confirmed using FT-IR, Ninhydrine’s test and Bradford protein assay. Physicochemical Properties were studied in vitro. Thrombolytic activity in vitro was determined using d–dimer indicator and weight of blood clot after treatment as indicators of thrombolytic activity. Results: Titanium nanoparticles show particle size at range 31 nm. The thrombolytic activity of streptokinase loading Titanium nanoparticles shows significant value in d-dimer and weight of blood clot compared with the control group and non-significant compared with an equivalent amount of streptokinase alone. Conclusion: Titanium nanoparticles conjugated with streptokinase show high thrombolytic activity against blood clots in vitro.

Abstract 17276: Tissue Plasminogen Activator Loaded Microbubbles for the Treatment of Coronary Microvascular Obstruction

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Stephen J D’Auria ◽  
Filip Istvanic ◽  
Francois Yu ◽  
Xucai Chen ◽  
John Pacella
Keyword(s):  
Enzymatic Activity ◽  
Microvascular Obstruction ◽  
Blood Clot ◽  
Bleeding Risk ◽  
Full Dose ◽  
Protein Assay ◽  
Loading Efficiency ◽  
Porcine Blood ◽  
Significant Difference

Introduction: Microvascular obstruction (MVO) occurs frequently during successful PCI for acute myocardial infarction. Our previous work has demonstrated the potential of using ultrasound targeted microbubbles (MBs) cavitation to relieve MVO, termed sonoreperfusion (SRP). Hypothesis: In the present study, we aim to develop a tPA loaded MB in order to utilize the thrombolytic effects of locally delivered tPA to enhance SRP, with a decreased tPA dose to limit the bleeding risk. Methods: tPA (1 mg/mL) was first modified via a maleimide linkage using BMCC-biotin, leading to a biotinylated protein. The biotinylated tPA (0.87 mg/mL) was then mixed with streptavidin labeled lipid shelled MB (1х10^9MB/mL) via biotin-streptavidin bridging. The enzymatic activity of the tPA in these tPA loaded MBs were was tested with whole porcine blood thrombus and with a tPA chromogenic activity kit. The loading capability of tPA onto the MBs was determined by BCA protein assay. The SRP efficacy of the loaded tPA MB was tested in an in vitro model of MVO and compared with full dose systemic tPA as well as modified MB. Results: tPA loaded MB showed successful lysis of whole porcine blood clot in vitro , as evidenced by decreased thrombus weight. Subsequent analysis of tPA activity showed that there was no significant difference in the activity of stock tPA when compared to the BMCC labeled tPA (42.0±3.4 IU vs 44.7±1.1 IU, p =0. 17). tPA was loaded onto MB at volumetric ratios of 3 units of tPA at 0.87 mg/mL to 1 unit (3:1) of MB at 1e9 MB/mL (43.5±2.2 IU, p =0.38) and 6:1 (39.3±1.5 IU, p =0.15). There was decreased activity in the 1:1 group. The loading capability of tPA for the 3:1 tPA loaded MB was 6.2e-7 μg per MB, with a loading efficiency of 10.4% of the stock tPA. Early in vitro work showed the tPA loaded MB to be non-inferior at relieving MVO with SRP when compared to infusion of full dose tPA and SRP with standard unmodified tPA. Conclusions: We successfully loaded tPA onto the surface of a lipid encapsulated MB, with retention of the enzymatic activity of tPA. The loading efficiency was 10.4% of the stock tPA. The tPA loaded MB demonstrated similar reperfusion efficacy to full systemic dose tPA. Thus, this approach of targeted local delivery of tPA may provide a means to mitigate bleeding risk without sacrificing drug efficacy.

PLATELET AGGREGATE-ASSOCIATED FIBRIN FORMATION AS A FUNCTION OF AGGREGATE SIZE AND HEPARIN CONCENTRATION

1987 ◽  
Author(s):  
L J Wurzinger ◽  
K Herbst ◽  
H Schmid-Schonbein
Keyword(s):  
Whole Blood ◽  
Aggregate Size ◽  
Arterial Disease ◽  
Amidolytic Activity ◽  
Protein Assay ◽  
Heparin Concentration ◽  
Platelet Aggregate ◽  
Minimum Number ◽  
Platelet Aggregates

The in vitro observation that fibrin forms withina few minutes in the crevices and niches inside and on the surface of platelet aggregates (PA), preparedfrom heparinized (5 U/ml) blood is consistent with the doubtful efficiency of heparin in the treatment of occlusive arterial disease (Thrcmb. Haemost. 46: 666,1981). Release of heparin- neutralizing proteins into limited and largely disclosed plasma compartments between aggregated platelets was held responsible for this remarkable phenomenon. However, the minimum number of aggregated platelets necessary to overcome the heparin inhibition remained undetermined then.PRP prepared from whole blood ant^coagulated with 0.5, 1 and 5 U/ml of mucosal heparin (Liquemin ), was aggregated with 10 or 100 pM ADP for 2 min at 37°C. Single PAs of various dimensions were withdrawn, washed, and incubated with a chromogenic substrate (S-2238, Kabi AB) to measure their thrombin content. Subsequently the number of platelets contained in the PA was evaluated by assaying the protein content of the aggregates. Microscopic PAs, their mass being toosmall to be determined precisely by a protein assay, were isolated with a filter technique, their extension was documented on photomicrographs for later calculation of aggregate volume and platelet content, before they were incubated with S-2238. Aggregates toosmall to develop detectable amidolytic activity, were checked microscopically for fibrin formed.S 2238 amidolytic activity (thrombin) in heparinizedPRP samples evolved as a linear function of the logarithm of PA mass. For a given heparin concentration (in whole blood) the following lowerthreshold platelet numbers of aggregates were found sufficient to allow the formation of detectable quantities of thrombin:These results suggest a fatal role platelet aggregates of minute dimensions may well play as a nidus of coagulation in fully heparinized blood.

scholarly journals Food protein: Food colour interactions and its application in rapid protein assay

2010 ◽  
Vol 28 (No. 6) ◽  
pp. 506-513 ◽  
Author(s):  
M.G. Saeed S ◽  
S.U. Abdullah ◽  
S.A. Sayeed ◽  
R. Ali
Keyword(s):  
Food Systems ◽  
Binding Capacity ◽  
Food Protein ◽  
Food Proteins ◽  
Protein Food ◽  
Peptide Bonds ◽  
Protein Assay ◽  
Food Dye ◽  
Almost All

The uniform distribution of colours as additives in a majority of the food systems is a reliable indication that one or more components of foods are able to bind with colour molecules and act as their carriers. However, the food components acting as the colour carriers have not been identified. The present paper describes the binding capacity of Carmoisine with a variety of food proteins, our results have shown that the intensity, staining, and sharpness of the stained protein bands were excellent as compared to Coomassie Brilliant Blue-R-250, which is an established staining agent for visualising electrophoretically resolved proteins. The data illustrates that Carmoisine is a fast reacting dye forming colour-complexes with all types of food proteins including curry leaves proteins. The protein bands are visualised within an hour which is useful for the initial immediate protein identifications. The experiments related to the staining of the resolved proteins with Carmoisine have shown that the dye is highly sensitive, rapid, and lasting. The food-dye can provide a quick protein assay as often desired in research works, the results may be later confirmed by using Coomassie if so required. In view of its strong binding with almost all proteins, it was thought that human proteases present in the digestive tract may not hydrolyse the bound proteins completely and may restrict the proteolytic digestion. However, the experiments based on the tryptic digestibility in vitro revealed that colour binding has no adverse effect on hydrolysis of peptide bonds by the intestinal proteases.

scholarly journals Analysis of Molecular Markers Genetically Linked to the Leptosphaeria maculans Avirulence Gene AvrLm1 in Field Populations Indicates a Highly Conserved Event Leading to Virulence on Rlm1 Genotypes

2002 ◽  
Vol 15 (7) ◽  
pp. 672-682 ◽  
Author(s):  
Agnès Attard ◽  
Lilian Gout ◽  
Mathieu Gourgues ◽  
Marie-Line Kühn ◽  
Jacques Schmit ◽  
...  
Keyword(s):  
Genetic Map ◽  
Large Scale ◽  
Polymerase Chain Reaction Amplification ◽  
Leptosphaeria Maculans ◽  
Avirulence Gene ◽  
Bac Contig ◽  
Genome Region ◽  
Repulsion Phase ◽  
Field Isolates

Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5′ end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race “AvrLm1” at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.

scholarly journals Structure and Synthesis of Antifungal Disulfide β-Strand Proteins from Filamentous Fungi

2018 ◽  
Vol 7 (1) ◽  
pp. 5 ◽  
Author(s):  
Györgyi Váradi ◽  
Gábor Tóth ◽  
Gyula Batta
Keyword(s):  
Filamentous Fungi ◽  
Disulfide Bonds ◽  
Antimicrobial Agents ◽  
Tertiary Structure ◽  
Fungal Infections ◽  
Recombinant Expression ◽  
Antifungal Protein ◽  
Significant Group ◽  
Antifungal Proteins ◽  
Neosartorya Fischeri

The discovery and understanding of the mode of action of new antimicrobial agents is extremely urgent, since fungal infections cause 1.5 million deaths annually. Antifungal peptides and proteins represent a significant group of compounds that are able to kill pathogenic fungi. Based on phylogenetic analyses the ascomycetous, cysteine-rich antifungal proteins can be divided into three different groups: Penicillium chrysogenum antifungal protein (PAF), Neosartorya fischeri antifungal protein 2 (NFAP2) and “bubble-proteins” (BP) produced, for example, by P. brevicompactum. They all dominantly have β-strand secondary structures that are stabilized by several disulfide bonds. The PAF group (AFP antifungal protein from Aspergillus giganteus, PAF and PAFB from P. chrysogenum, Neosartorya fischeri antifungal protein (NFAP)) is the best characterized with their common β-barrel tertiary structure. These proteins and variants can efficiently be obtained either from fungi production or by recombinant expression. However, chemical synthesis may be a complementary aid for preparing unusual modifications, e.g., the incorporation of non-coded amino acids, fluorophores, or even unnatural disulfide bonds. Synthetic variants up to ca. 6–7 kDa can also be put to good use for corroborating structure determination. A short overview of the structural peculiarities of antifungal β-strand disulfide bridged proteins will be given. Here, we describe the structural propensities of some known antifungal proteins from filamentous fungi which can also be prepared with modern synthetic chemistry methods.

Serum Proteins Opsonization and Phagocytic Uptake of PEG-Modified PLGA Nanoparticles: Effect of Particle Size

2011 ◽  
Vol 393-395 ◽  
pp. 939-942 ◽  
Author(s):  
An Shu Yang ◽  
Wei Liu ◽  
Xiang Liang Yang
Keyword(s):  
Particle Size ◽  
Serum Protein ◽  
Complement Activation ◽  
Serum Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spontaneous Emulsification ◽  
Protein Assay ◽  
Effect Of Particle Size ◽  
Serum Dependent

The purpose of this study is to evaluate the effect of particle size on serum protein opsonization and in vitro macrophage uptake of polyethyleneglycol modified poly (D, L-lactide-co-glycolide) nanoparticles (PEG-PLGA-NPs). PEG-PLGA-NPs were prepared by modified-spontaneous emulsification solvent diffusion (modified-SESD) method. Serum protein adsorptions to PEG-PLGA-NPs were evaluated by bicinchoninic acid (BCA) protein assay and enzyme-linked immunosorbent assay (ELISA). Complement activation was also investigated by ELISA for complement fragments iC3b. Uptake of PEG-PLGA-NPs by macrophages was measured by fluorescence spectrometer. The results showed that serum protein adsorption and complement activation were augmented for nanoparticles with a larger size below 400 nm. Phagocytosis of PEG-PLGA-NPs by murine peritoneal macrophages involved serum-independent and serum-dependent phagocytosis. Serum-independent phagocytosis decreased, while serum-dependent phagocytosis increased with the increase of particle size in the nanometer and submicrometer range. Consequently, nanoparticles with size of about 400 nm were phagocytosed more readily than either smaller or larger particles

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