Electrophoretic analysis of effects of in vitro heating on proteins and enzymes from dormant peanuts (Arachis hypogaea)

1973 ◽  
Vol 51 (6) ◽  
pp. 1191-1196 ◽  
Author(s):  
Dempsey L. Thomas ◽  
James E. Bright
Keyword(s):  
Thermal Sensitivity ◽  
Distribution Patterns ◽  
Electrophoretic Analysis ◽  
Staining Intensity ◽  
Soluble Proteins ◽  
Starch Gel Electrophoresis ◽  
Complete Inactivation ◽  
Reserve Protein ◽  
Different Temperatures

Protein extracts from dormant peanut cotyledons were heated to determine the influence of different temperatures on banding characteristics of soluble proteins and enzymes by use of horizontal starch gel electrophoresis. Soluble proteins showed no significant change between 25° and 85 °C. At 95 °C, however, major bands of reserve protein decreased in staining intensity and new bands with greater migration velocity appeared; banding still occurred at 100 °C. Distribution patterns of glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), nonspecific α-esterases (α-EST), and leucine aminopeptidase (LAP) reflected no change in banding or apparent enzyme activity at temperatures less than 45 °C. Complete inactivation was estimated for GDH at 80 to 85 °C, MDH at 65 °C, α-EST at 60 to 65 °C, and LAP at 60 to 65 °C. Isoenzymes of GDH, MDH, and LAP, and bands of α-EST exhibited differential thermal sensitivity. Peroxidase activity (using pyrogallol as the hydrogen donor) was not influenced through 65 °C; minimal reaction with benzidine occurred. No alkaline phosphatase activity was observed. Banding patterns of proteins or enzymes heated for 15 min were identical with those heated for 30 min. The results are compared with those of other studies regarding the influence of heat on proteins and enzymes in extracts of heated cotyledons from 5-day peanut seedlings and in extracts from wet- and dry-roasted seed.

Effects of juvenile hormone, ecdysterone, actinomycin D, and mitomycin C on the cuticular proteins of Tenebrio molitor

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 107-123
Author(s):  
P. Elaine Roberts ◽  
Judith H. Willis
Keyword(s):  
Juvenile Hormone ◽  
Mitomycin C ◽  
Actinomycin D ◽  
Electrophoretic Analysis ◽  
Tenebrio Molitor ◽  
Staining Intensity ◽  
Soluble Proteins ◽  
Blood Proteins ◽  
Intense Staining ◽  
Cuticular Proteins

Juvenile hormone (JH), ecdysterone and some antibiotics cause Tenebrio molitor to form a second pupa or pupal-adult intermediate. Incorporation of labelled leucine into the cuticular proteins of JH-induced second pupae did not differ from incorporation in normal pupae, and the soluble cuticular proteins from these young second pupae were identical to those extracted from normal pupal exocuticle when analysed by SDS-polyacry lamide gel electrophoresis. However, as these second pupae aged, the major early bands did not undergo a normal decrease in staining intensity, indicating a JH effect on protein insolubilization (sclerotization). The transport of protein into the cuticle may also have been altered by JH; electrophoretic analysis of the new cuticle of treated animals showed intense staining of bands with RF'S similar to those of blood proteins. The new exocuticle produced after treatment of pupae with ecdysterone had soluble proteins which were typical of normal pupae, but extracts from such animals aged prior to cuticle removal yielded bands characteristic of normal adults. Pupae treated with actinomycin D occasionally form new abdominal cuticle with characteristic pupal morphology. These cuticles yielded soluble proteins which, upon analysis, had pupal, pupal and adult, or adult banding patterns. Animals treated with mitomycin C, although retaining vestiges of pupal abdominal characters, had adult cuticular proteins.

Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

2019 ◽  
Vol 35 (6) ◽  
pp. 91-101
Author(s):  
F.A. Klebanov ◽  
S.E. Cheperegin ◽  
D.G. Kozlov
Keyword(s):  
Penicillium Chrysogenum ◽  
Protein Denaturation ◽  
Biologically Active ◽  
Cultivation Temperature ◽  
Target Proteins ◽  
E Coli ◽  
Complete Inactivation ◽  
Target Molecules ◽  
Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

scholarly journals Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aiyan Guan ◽  
Inge Van Damme ◽  
Frank Devlieghere ◽  
Sarah Gabriël
Keyword(s):  
Enzymatic Digestion ◽  
Infected Fish ◽  
Effect Of Temperature ◽  
Video Recordings ◽  
Different Temperatures ◽  
Semi Solid ◽  
Zoonotic Parasites ◽  
The Impact ◽  
Phosphate Buffered Saline

AbstractAnisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.

scholarly journals Efficiency of Antimicrobial Photodynamic Therapy with Photodithazine® on MSSA and MRSA Strains

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 869
Author(s):  
Beatriz Müller Nunes Souza ◽  
Juliana Guerra Pinto ◽  
André Henrique Correia Pereira ◽  
Alejandro Guillermo Miñán ◽  
Juliana Ferreira-Strixino
Keyword(s):  
Staphylococcus Aureus ◽  
Photodynamic Therapy ◽  
Antimicrobial Photodynamic Therapy ◽  
Bacterial Inactivation ◽  
Bacterial Reduction ◽  
Complete Inactivation ◽  
Opportunistic Bacteria ◽  
Significant Difference ◽  
Mrsa Strains

Staphylococccus aureus is a ubiquitous and opportunistic bacteria associated with high mortality rates. Antimicrobial photodynamic therapy (aPDT) is based on the application of a light source and a photosensitizer that can interact with molecular oxygen, forming Reactive Oxygen Species (ROS) that result in bacterial inactivation. This study aimed to analyze, in vitro, the action of aPDT with Photodithazine® (PDZ) in methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains. The strains were incubated with PDZ at 25, 50, 75, and 100 mg/L for 15 min and irradiated with fluences of 25, 50, and 100 J/cm2. The internalization of PDZ was evaluated by confocal microscopy, the bacterial growth by counting the number of colony-forming units, as well as the bacterial metabolic activity post-aPDT and the production of ROS. In both strains, the photosensitizer was internalized; the production of ROS increased when the aPDT was applied; there was a bacterial reduction compared to the control at all the evaluated fluences and concentrations; and, in most parameters, it was obtained complete inactivation with significant difference (p < 0.05). The implementation of aPDT with PDZ in clinical strains of S. aureus has resulted in its complete inactivation, including the MRSA strains.

scholarly journals Chromatography-Independent Fractionation and Newly Identified Molecular Features of the Adzuki Bean (Vigna angularis Willd.) β-vignin Protein

2021 ◽  
Vol 22 (6) ◽  
pp. 3018
Author(s):  
Biane Philadelpho ◽  
Victória Souza ◽  
Fabiani Souza ◽  
Johnnie Santos ◽  
Fabiana Batista ◽  
...  
Keyword(s):  
Metal Binding ◽  
Cardiovascular Health ◽  
Binding Capacity ◽  
Biological Activities ◽  
Electrophoretic Analysis ◽  
Adzuki Bean ◽  
Molecular Features ◽  
Health Promoting ◽  
High Degree

Adzuki seed β-vignin, a vicilin-like globulin, has proven to exert various health-promoting biological activities, notably in cardiovascular health. A simple scalable enrichment procedure of this protein for further nutritional and functional studies is crucial. In this study, a simplified chromatography-independent protein fractionation procedure has been optimized and described. The electrophoretic analysis showed a high degree of homogeneity of β-vignin isolate. Furthermore, the molecular features of the purified protein were investigated. The adzuki bean β-vignin was found to have a native size of 146 kDa, and the molecular weight determined was consistent with a trimeric structure. These were identified in two main polypeptide chains (masses of 56–54 kDa) that are glycosylated polypeptides with metal binding capacity, and one minor polypeptide chain with a mass 37 kDa, wherein these features are absent. The in vitro analysis showed a high degree of digestibility of the protein (92%) and potential anti-inflammatory capacity. The results lay the basis not only for further investigation of the health-promoting properties of the adzuki bean β-vignin protein, but also for a possible application as nutraceutical molecule.

scholarly journals Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1484
Author(s):  
Felice Panebianco ◽  
Selene Rubiola ◽  
Francesco Chiesa ◽  
Tiziana Civera ◽  
Pierluigi Aldo Di Ciccio
Keyword(s):  
Listeria Monocytogenes ◽  
In Vitro Study ◽  
Planktonic Cells ◽  
Free Living ◽  
Biofilm Inhibition ◽  
Additional Tool ◽  
Complete Inactivation ◽  
Gaseous Ozone ◽  
Food Borne

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

Thermal sensitivity of Bullfrog's immune response kept at different temperatures

2020 ◽  
Author(s):  
Alan S. Lima ◽  
Letícia de F. Ferreira ◽  
Diego P. Silva ◽  
Fernando R. Gomes ◽  
Stefanny C. M. Titon
Keyword(s):  
Immune Response ◽  
Thermal Sensitivity ◽  
Different Temperatures

Bacteriophage-host depth distribution patterns in soil are maintained after nutrient stimulation in vitro

2021 ◽  
Vol 787 ◽  
pp. 147589
Author(s):  
Xiaolong Liang ◽  
Yusong Wang ◽  
Ying Zhang ◽  
Bingxue Li ◽  
Mark Radosevich
Keyword(s):  
Depth Distribution ◽  
Distribution Patterns

scholarly journals Complete Inactivation of Blood Borne Pathogen Trypanosoma cruzi in Stored Human Platelet Concentrates and Plasma Treated With 405 nm Violet-Blue Light

2020 ◽  
Vol 7 ◽  
Author(s):  
Katarzyna I. Jankowska ◽  
Rana Nagarkatti ◽  
Nirmallya Acharyya ◽  
Neetu Dahiya ◽  
Caitlin F. Stewart ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Human Plasma ◽  
Blue Light ◽  
Chagas Disease ◽  
Light Treatment ◽  
Platelet Concentrates ◽  
Complete Inactivation ◽  
Pathogen Reduction ◽  
Biochemical Indices

The introduction of pathogen reduction technologies (PRTs) to inactivate bacteria, viruses and parasites in donated blood components stored for transfusion adds to the existing arsenal toward reducing the risk of transfusion-transmitted infectious diseases (TTIDs). We have previously demonstrated that 405 nm violet-blue light effectively reduces blood-borne bacteria in stored human plasma and platelet concentrates. In this report, we investigated the microbicidal effect of 405 nm light on one important bloodborne parasite Trypanosoma cruzi that causes Chagas disease in humans. Our results demonstrated that a light irradiance at 15 mWcm−2 for 5 h, equivalent to 270 Jcm−2, effectively inactivated T. cruzi by over 9.0 Log10, in plasma and platelets that were evaluated by a MK2 cell infectivity assay. Giemsa stained T. cruzi infected MK2 cells showed that the light-treated parasites in plasma and platelets were deficient in infecting MK2 cells and did not differentiate further into intracellular amastigotes unlike the untreated parasites. The light-treated and untreated parasite samples were then evaluated for any residual infectivity by injecting the treated parasites into Swiss Webster mice, which did not develop infection even after the animals were immunosuppressed, further demonstrating that the light treatment was completely effective for inactivation of the parasite; the light-treated platelets had similar in vitro metabolic and biochemical indices to that of untreated platelets. Overall, these results provide a proof of concept toward developing 405 nm light treatment as a pathogen reduction technology (PRT) to enhance the safety of stored human plasma and platelet concentrates from bloodborne T. cruzi, which causes Chagas disease.

scholarly journals Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2.

1991 ◽  
Vol 115 (5) ◽  
pp. 1345-1356 ◽  
Author(s):  
B E Flucher ◽  
J L Phillips ◽  
J A Powell
Keyword(s):  
Muscle Cells ◽  
Distribution Patterns ◽  
Receptor Alpha ◽  
Alpha Subunits ◽  
Nuclear Domain ◽  
T Tubules ◽  
Immunofluorescence Labeling ◽  
T System ◽  
Triad Junctions

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.

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