Sodium Dodecyl Sulfate-polyacrylamide Gel Research Articles

Coprecipitated Keratin/hydroxyapatite nanocomposites assisted by microwave/ultrasound irradiation, and its cytotoxic evaluation on NIH/3T3 fibroblast cells

This work is aimed to synthesize Hydroxyapatite (Hap) and Keratin/Hydroxyapatite (KerHap) nanocomposites by coprecipitation using the simultaneous microwave/ultrasound (Mw/Us) method to confer specific textural and morphological properties to the materials using keratin (Ker) extracted from human hair. Four materials were synthesized applying different powers of Mw/Us irradiation: Hap (1100 W Mw, 40 kHz Us), KerHap A (700 W Mw, 40 kHz Us), KerHap B (900 W Mw, 40 kHz Us) and KerHap C (1100 W Mw, 40 kHz Us) at 120 °C for 10 min. The materials were characterized according to their structure (FTIR, XRD, Raman spectroscopy), morphology and size (TEM), and thermal properties (TG). Their cytotoxic effects were studied at different concentrations (25, 50, 100, 200, and 400 μg/mL) using the fibroblast cell line (NIH/3T3) by MTT assay at 24, 48, and 72 h. The Ker extract exhibited a major molecular weight band between 11–10 kDa in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Morphology, particle, and crystallite sizes were dependent on the Mw irradiation power applied and keratin added during the synthesis. The MTT assay at 48 h demonstrated a significant increase in cell viability of 39% for Hap at 50 µg/mL, for KerHap A did not show a significant difference, while KerHap B-C showed a significant increase at 200 µg/mL of 30% in cell viability. It means that fibroblast presented better cell adhesion on Hap than on KerHap A-C composites; it may be due to keratin blocking Hap pores because of electrostatic interactions between keratin and Hap.

Purification and characterization of α-galactosidase isolated from Klebsiella pneumoniae in the human oral cavity

The enzyme α-Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa.

The structural characteristics and physicochemical properties of mung bean protein hydrolysate of protamex induced by ultrasound.

The limited physicochemical properties (such as low foaming and emulsifying capacity) of mung bean protein hydrolysate restrict its application in the food industry. Ultrasound treatment could change the structures of protein hydrolysate to accordingly affect its physicochemical properties. The aim of this study was to investigate the effects of ultrasound treatment on the structural and physicochemical properties of mung bean protein hydrolysate of protamex (MBHP). The structural characteristics of MBHP were evaluated using tricine sodium dodecylsulfate-polyacrylamide gel electrophoresis, laser scattering, fluorescence spectrometry, etc. Solubility, fat absorption capacity and foaming, emulsifying and thermal properties were determined to characterize the physicochemical properties of MBHP. MBHP and ultrasonicated-MBHPs (UT-MBHPs) all contained five main bands of 25.8, 12.1, 5.6, 4.8 and 3.9 kDa, illustrating that ultrasound did not change the subunits of MBHP. Ultrasound treatment increased the contents of α-helix, β-sheet and random coil and enhanced the intrinsic fluorescence intensity of MBHP, but decreased the content of β-turn, which demonstrated that ultrasound modified the secondary and tertiary structures of MBHP. UT-MBHPs exhibited higher solubility, foaming capacity and emulsifying properties than MBHP, among which MBHP-330 W had the highest solubility (97.32%), foaming capacity (200%), emulsification activity index (306.96 m2 g-1 ) and emulsion stability index (94.80%) at pH 9.0. Ultrasound treatment enhanced the physicochemical properties of MBHP, which could broaden its application as a vital ingredient in the food industry. © 2023 Society of Chemical Industry.

Physicochemical Features and Volatile Organic Compounds of Horse Loin Subjected to Sous-Vide Cooking.

The purpose of this study was to evaluate the effect of temperature and time of sous-vide cooking method on the characteristics of Thoroughbred horse loin. Sliced portions (200 ± 50 g) were cooked by boiling (control) and sous-vide (65 and 70 °C for 12, 18, and 24 h). The samples were analyzed for proximate composition, pH, color, texture, microstructure, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), microbiology, volatile organic compounds (VOCs), nucleotide content, and fatty acids composition. The color analysis showed decreased redness at elevated temperatures. Improved tenderness, demonstrated by reduced shear force values (36.36 N at 65 °C for 24 h and 35.70 N at 70 °C for 24 h). The micrographs indicated dense fiber arrangements at 70 °C. The SDS-PAGE revealed muscle protein degradation with extended sous-vide cooking. The VOC analysis identified specific compounds, potentially distinctive markers for sous-vide cooking of horse meat including 1-octen-3-ol, decanal, n-caproic acid vinyl ester, cyclotetrasiloxane, octamethyl, and 3,3-dimethyl-1,2-epoxybutane. This study highlights the cooking time's primary role in sous vide-cooked horse meat tenderness and proposes specific VOCs as potential markers. Further research should explore the exclusivity of these VOCs to sous-vide cooking.

The process of solid-state fermentation of soybean meal: antimicrobial activity, fermentation heat generation and nitrogen solubility index.

Bacillus amyloliquefaciens has excellent protease production ability and holds great prospects for application in the solid-state fermentation of soybean meal (SBM). Among eight strains of bacteria, Bacillus amyloliquefaciens subsp. plantarum CICC 10265, which exhibited higher protease production, was selected as the fermentation strain. The protease activity secreted by this strain reached 106.41 U mL-1 . The microbial community structure differed significantly between natural fermentation and inoculation-enhanced fermented soybean meal (FSBM), with the latter showing greater stability and inhibition of miscellaneous bacterial growth. During fermentation, the temperature inside the soybean meal increased, and the optimal environmental temperature for FSBM was found to be between 35 and 40 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and nitrogen solubility index (NSI) results demonstrated that solid-state fermentation had a degrading effect on highly denatured proteins in SBM, resulting in an NSI of 67.1%. Bacillus amyloliquefaciens subsp. plantarum CICC 10265 can enhance the NSI of SBM in solid-state fermentation and inhibit the growth of miscellaneous bacteria. © 2023 Society of Chemical Industry.

In vitro assessment of thrombolytic potential of red and white ginger (Zingiber officinale)

Background: Cardiovascular diseases (CVDs) are a leading cause of death, and their pathogenesis is commonly attributed to thrombosis. Although existing medications are effective and fast-acting for thrombosis management, they tend to be expensive and cause severe side effects. Plant-based thrombolytic agents are actively sought after as inexpensive and safe alternatives for the treatment and prevention of thrombosis. Red ginger (Zingiber officinale var. rubrum) and white ginger (Zingiber officinale var. officinale) are widely used in foods and beverages and are believed to confer a wide variety of health benefits. Objective: This study aims to elucidate the thrombolytic and fibrinolytic potential of red and white ginger extracts in vitro.Methods: In this study, in vitro analyses were performed using erythrocyte liberation, euglobulin degradation, fibrin degradation, and fibrin zymography assays. The ability of crude enzyme extracts from both red (rgEx) and white ginger (wgEx) to degrade blood clots was analyzed using the erythrocytes liberation assay. Then, the thrombolytic and fibrinolytic activities of rgEx and wgEx were evaluated using euglobulin and fibrin degradation assays, both of which were visualized using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Finally, fibrinolytic enzymes were identified using a fibrin zymography assay. Results: Red and white ginger extracts were found to have strong thrombolytic properties via high total liberated erythrocyte count from the erythrocyte liberation assay. The ginger extract proteases can rapidly degrade euglobulin and fibrin, with their priority order beginning with A

Physicochemical and biological stability of diluted vedolizumab in intravenous infusion bags

IntroductionIntravenous vedolizumab is a widely used monoclonal antibody for outpatients with inflammatory bowel disease. Drug preparation is performed on the day of administration, but is time consuming, causing unnecessary in-hospital...

The Blood Group Rhc Protein from Human Erythrocyte Membranes as an Immunogen for Producing Antibodies in Mice

Background: Various methods have been used to isolate red blood cell (RBC) membrane antigens. In this regard, obtaining the antigen and preserving its structure is of special importance. However, limited studies have been conducted to purify cellular membrane antigens such as Rh proteins.
 Materials and Methods: In this experimental study, Rhc antigens of the RBC membrane was purified. Here, the RBC membrane was solubilized through the lysis buffer. Next, dialysis and affinity chromatography were performed using polyclonal anti-human RhCcEe antibody to isolate Rhc/e antigens from the RBCs with the following blood group characteristics: Rhc+, RhC-, Rhe+, and RhE-. The purified proteins were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot methods. The immunization process was performed in Balb/c mice using the Rhc antigen as an immunogen. After the last injection, the mouse serum was used to titrate antibodies.
 Results: Protein bands of the purified antigen were observed in the silver-stained SDS-PAGE gel (region of 25-35 kDa). The OD405nm = 0.56 ± 0.05 results showed the reactivity with Rhc antibody. The specificity of the purified protein was evaluated using the dot blot assay. The anti-sera titration was greater than 1/10,000 against Rhc-coated microwells. Rh antigens can be isolated from the RBC membrane using the non-ionic NP-40 detergent and affinity chromatography.
 Conclusion: The Rh antigen can be isolated from the RBC membrane with proper purity by solubilization with the non-ionic NP-40 detergent and purification by affinity chromatography. It seems that the membrane antigen maintains its antigenicity and structure. As a result, it can be detected by blood group-specific antibodies used in the hemagglutination method. Purified antigens may be used to generate antibodies or to study the protein structure.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents

We previously reported that bakuchiol, a phenolic isoprenoid anti-cancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney (MDCK) cell lysates and purified the specific bakuchiol-binding proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using nano-liquid chromatography with tandem mass spectrometry analysis, we identified prohibitin (PHB) 2, voltage dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and co-localized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein (NP) expression levels and viral titers in the conditioned medium of virus-infected MDCK cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Effect of dynamic high-pressure microfluidization treatment on soybean protein isolate–rutin non-covalent complexes

In this investigation, soybean protein isolate–rutin (SPI–RT) complexes were treated using dynamic high-pressure microfluidization (DHPM). The effects of this process on the physicochemical and thermodynamic properties of SPI were investigated at different pressures. Fourier-transform infrared spectroscopy and fluorescence spectroscopy provided evidence that the SPI structure had been altered. The binding of SPI to RT resulted in a decrease in the percentage of α-helices and random curls as well as an increase in the percentage of β-sheets. In particular, the α-helix content decreased from 29.84 % to 26.46 %, the random curl content decreased from 17.45 % to 15.57 %, and the β-sheet content increased from 25.37 % to 26.53 %. Moreover, fluorescence intensity decreased, and the emission peak of the complex was red-shifted by 6 nm, exposing the internal groups. Based on fluorescence quenching analysis, optimal SPI–RT complexation was achieved after 120-MPa DHPM treatment, and molecular docking analysis verified the interaction between SPI and RT. The minimum particle size, maximum absolute potential, and total phenolic content of the complexes were 78.06 nm, 21.4 mV and 74.35 nmol/mg protein, respectively. Furthermore, laser confocal microscopy revealed that the complex particles had the best microstructure. Non-covalent interactions between the two were confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Moreover, the hydrophobicity of the complex particle's surface increased to 16,045 after 120-MPa DHPM treatment. The results of this study suggest that DHPM strongly promotes the improvement of the physicochemical properties of SPI, and provide a theoretical groundwork for further research.

Growth and protein response of rice plant with plant growth-promoting rhizobacteria inoculations under salt stress conditions.

Soil salinity has been one of the significant barriers to improving rice production and quality. According to reports, Bacillus spp. can be utilized to boost plant development in saline soil, although the molecular mechanisms behind the interaction of microbes towards salt stress are not fully known. Variations in rice plant protein expression in response to salt stress and plant growth-promoting rhizobacteria (PGPR) inoculations were investigated using a proteomic method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Findings revealed that 54 salt-responsive proteins were identified by mass spectrometry analysis (LC-MS/MS) with the Bacillus spp. interaction, and the proteins were functionally classified as gene ontology. The initial study showed that all proteins were labeled by mass spectrometry analysis (LC-MS/MS) with Bacillus spp. interaction; the proteins were functionally classified into six groups. Approximately 18 identified proteins (up-regulated, 13; down-regulated, 5) were involved in the photosynthetic process. An increase in the expression of eight up-regulated and two down-regulated proteins in protein synthesis known as chaperones, such as the 60kDa chaperonin, the 70kDa heat shock protein BIP, and calreticulin, was involved in rice plant stress tolerance. Several proteins involved in protein metabolism and signaling pathways also experienced significant changes in their expression. The results revealed that phytohormones regulated the manifestation of various chaperones and protein abundance and that protein synthesis played a significant role in regulating salt stress. This study also described how chaperones regulate rice salt stress, their different subcellular localizations, and the activity of chaperones.

Effects of Flavourzyme and Alkaline Protease Treatment on Structure and Allergenicity of Peanut Allergen Ara h 1.

Peanut allergy poses a significant threat to human health due to the increased risk of long-term morbidity at low doses. Modifying protein structure to affect sensitization is a popular topic. In this study, the purified peanut allergen Ara h 1 was enzymatically hydrolysed using Flavourzyme, alkaline protease or a combination of both. The binding ability of Ara h 1 to antibodies, gene expression and secretion levels of the proinflammatory factors interleukin-5 and interleukin-6 in Caco-2 cells was measured. Changes in the secondary and tertiary structures before and after treatment with Ara h 1 were analysed by circular dichroism and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated a decrease of the allergenicity and proinflammatory ability of Ara h 1. The evaluation showed that the Flavourzyme and alkaline protease treatments caused particle shortening and aggregation. The fluorescence emission peak increased by 3.4-fold after the combined treatment with both proteases. Additionally, the secondary structure underwent changes and the hydrophobicity also increased 8.95-fold after the combined treatment. These findings partially uncover the mechanism of peanut sensitization and provide an effective theoretical basis for the development of a new method of peanut desensitization.

Determination of genetic diversity in Acacia modesta germplasm using SDS-PAGE.

Biochemical markers such as protein are very important to determine genetic diversity among plant species in a given population which in turn is very important for breeders and farmers as they can then easily select the most appropriate variety to grow in a given locality. In this connection, the present study is aimed to evaluate genetic diversity in Acacia modesta germplasm through Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) technique. About 40 genotypes were subjected to SDS-PAGE analysis where a total of 12 polypeptide bands were observed in electrophoretogram. Out of which 16.67% were monomorphic while the remaining 83.33% were polymorphic. Variation found in B-2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, were 20, 22.50, 32.50, 10, 2.50, 22.50, 15, 5, 2.50 and 75% respectively. Locus contribution toward genetic disagreement was 83.33%. Cluster analysis sorted all the genotypes into 9 clusters. The genotypes in one cluster were identical regarding protein profiling and showed less intra-specific genetic variation whereas differences were find from other genotypes.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2.

In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Protein profiles of follicle fluid of different sizes in cows and buffaloes

In vitro embryo production systems in buffaloes have great productive perspectives and opportunities for improvement. Among these, comparative studies with species with more significant advances in reproductive biotechnology have been developed. Accordingly, this work aimed to identify the differences in the electrophoretic profiles of proteins in the follicular fluid (FF) of cows and buffaloes and their possible association with follicle size. FF was obtained at the central abattoir in Medellín (Antioquia), Colombia, from small (<7 mm) and large (>7 mm) follicles from the ovaries of 25 cows (Bos indicus) and 20 buffaloes (Bubalus bubalis). The total protein content of the FF was quantified and subsequently depleted of albumin and immunoglobulins. Samples were subjected to denaturing electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) to determine the electrophoretic profiles using a photodocumenter. The values obtained for the relative amount of each band were compared between species and follicle sizes using the Mann-Whitney test. The results showed no significant differences in total protein concentration between the different follicle sizes and species. Further, 72.6% of the FF proteins are immunoglobulins and albumin. The profiles of small follicles (<7mm) in cows presented 19 bands and 11 in buffaloes. The molecular weight range of the bands detected was between 5 and 250 kDa. Quantitative differences of the proteins in the follicular fluids evaluated were identified. The information obtained may contribute to elucidating the physiological differences between large and small follicles but does not explain the differences between species.

Zymography of proteases in honey bees (Apis Mellifera) infected with Nosema ceranae

Nosemosis is one of the most important honey bee diseases and is caused by two fungal species of the genus Nosema, i.e., Nosema apis and Nosema ceranae. Evidence suggests that in the coming years, Nosema ceranae, which is less sensitive to fumagillin, could replace Nosema apis as the predominant pathogenic Nosema. Identifying and developing drugs that target these microsporidia without having serious effects on host bee physiology is essential for controlling nosemosis, and this requires information about bee enzymes. Serine proteases, the major protease enzymes in insects, play an important role in the insect immune system. In the present study, some proteases of honey bees infected with N. ceranae were studied by zymography. PCR tests showed that all honey bee samples were infected with N. ceranae. Also, the protein pattern of infected honey bee tissue extracts was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands with molecular weights of 25, 35, 48, 63, 75, and 100 kDa were observed. Subsequently, the serine proteases in the bees? digestive tracts ranged in molecular weight between 23-35 kDa, and the molecular weight of cathepsins was 25 kDa by gelatin zymography. Investigation of bee proteases might be useful for acquiring more knowledge about the interaction between host and parasite and to access an effective and low-risk compound for nosemosis treatment.

Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 μg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Extração e quantificação de anticorpos entivenenos em ovos de galinha contra veneno de escorpião

ABSTRACT: Avian-derived IgY is thought to be the best therapy for scorpion bites concerning low-level side effects. The present study analyzed a hypothesis about the neutralization of scorpion venom Androcotonus australis through antibodies produced in the egg yolks of chickens. The venom used for inoculation was obtained from Androctonus australis (yellow fat-tailed scorpion) from southern Punjab, Pakistan. The lethal dose of LD50 against scorpion venom was calculated in chickens and mice. Safe doses were given to egg-laying chickens to produce IgY antibodies. The antivenom IgY antibodies were extracted from the egg yolks of immunized chicken using the polyethylene glycol (PEG) method. Moreover, IgY was confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Ouchterlony double immunodiffusion assay test. The antibody titers were evaluated by the enzyme-linked immunosorbent assay (ELISA). The neutralisation capacity of extracted anti-scorpion antibodies was tested on mice. The calculated LD50 of scorpion venom for chicken and mice was 4 mg/kg and 2.5 mg/kg, respectively. SDS-PAGE and Ouchterlony double immunodiffusion confirmed the presence of IgY against scorpion venom. The maximum titer value of specific IgY produced against scorpion venom was 3.5 ug/ml. A concentration of 220 ul/LD50 was effective to neutralize 1 mg of scorpion venom. It is suggested that IgY obtained from egg yolks is safe against targeted venom and can be used as an effective alternative to equine IgG antibodies against scorpion envenoming.