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Cleavage Of Structural Proteins During Assembly Of Head Of Bacteriophage-T4
Abstract
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
... To ensure that the obtained molecule corresponds to the same molecule as the methodology proposed by Carvalho (Carvalho et al., 2016), characterization was carried out through antitryptic activity (Kakade et al., 1969); protein quantification (Bradford, 1976), and discontinuous and denaturing polyacrylamide gel (SDS-PAGE) (Laemmli, 1970). ...
... This methodology was performed according to Laemmli (Laemmli, 1970) to determine the degree of purity and estimate the molecular mass of the proteins contained in CE, F1, F2, and TTI, for which a 12 % SDS-PAGE was performed. To measure the molecular weight of the proteins, 5 µL of Amersham ECL Rainbow Marker -High range (Sigma-Aldrich) was used as a parameter, which contains different molecular masses (225 kDa, 76 kDa, 52 kDa, 38 kDa, 31 kDa, 24 kDa, 17 kDa, and 12 kDa) and aliquots containing 10 µL of CE, F1 and F2, and 5 µL and 10 µL of proteins for TTI. ...
... Sterilized distilled water was used as a control (without TTI addition), being submitted to the same conditions as the TTI (Fig. 2). During the gastric and intestinal phases, aliquots were taken at 20, 60, and 120 min for later analysis of the monitoring of the TTI hydrolysis pattern through the 15 % SDS-PAGE gel (Laemmli, 1970). Aliquots of the oral phase were not taken because there is no protein hydrolysis in this phase. ...
The aim of this study was to prospect in silico peptides derived from a multifunctional protein and assess their interaction with the insulin receptor (IR). The trypsin inhibitor isolated from tamarind seeds (TTI) was obtained through trypsin-sepharose 4B-CNBr affinity chromatography and subsequently characterized. The TTI underwent in vitro hydrolysis to assess its susceptibility to enzymatic degradation and determine suitable enzymes for cleavage in silico. The theoretical model was established to assess the purified tamarind seed trypsin inhibitor (TTIp 56/287) being cleaved in silico and selected for simulation by molecular dynamics. Among the peptides generated, Peptidetripquimo59 presented the most negative docking score (-175.53) with the IR, indicating strong affinity and stability in complex formation. Significant interaction with the IR was observed for key residues, including arginine 16 (-209.07 kJ mol-1), threonine 1 (-148.54 kJ mol-1), and valine 2 (-94.53 kJ mol-1). Additionally, it was discovered that both insulin and Peptidetripquimo59 exhibit binding to the identical location on the insulin receptor (IR). The results of the semi-empirical approach revealed that Peptide-tripquimo59 exhibited greater potential for interaction with the IR compared to other complexes such as the insulin-IR complex, suggesting its candidacy as a starting point for the development of therapeutic agents targeting both type 1 and type 2 Diabetes mellitus.
... For an initial expression check, single colonies were used to inoculate 1 mL of Overnight Express (Millipore-Sigma, Billerica, USA) which was shaken overnight at 37 °C. A sample (250 μL) was subjected to heat denaturation and analyzed by SDS-PAGE (Laemmli 1970). ...
Daily agro-industrial waste, primarily cellulose, lignin, and hemicellulose, poses a significant environmental challenge. Harnessing lignocellulolytic enzymes, particularly endo-1,4-β-xylanases, for efficient saccharification is a cost-effective strategy, transforming biomass into high-value products. This study focuses on the cloning, expression, site-directed mutagenesis, purification, three-dimensional modeling, and characterization of the recombinant endo-1,4-β-xylanase (XlnA) from Aspergillus clavatus in Escherichia coli. This work includes evaluation of the stability at varied NaCl concentrations, determining kinetic constants, and presenting the heterologous expression of XlnAΔ36 using pET22b(+). The expression led to purified enzymes with robust stability across diverse pH levels, exceptional thermostability at 50 °C, and 96–100% relative stability after 24 h in 3.0 M NaCl. Three-dimensional modeling reveals a GH11 architecture with catalytic residues Glu 132 and 22. XlnAΔ36 demonstrates outstanding kinetic parameters compared to other endo-1,4-β-xylanases, indicating its potential for industrial enzymatic cocktails, enhancing saccharification. Moreover, its ability to yield high-value compounds, such as sugars, suggests a promising and ecologically positive alternative for the food and biotechnology industries.
... SDS-PAGE was performed using a 5% stacking gel and 10% resolving gel under reducing conditions, as described by Laemmli (Laemmli 1970;Varol et al. 2023). Protein concentration was determined using a Bio-Rad protein assay kit and bovine serum albumin as the standard (Bradford 1976). ...
The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level of the recombinant enzyme was 10.7-fold higher than the expression level of the native one (0.13 mg mL −1). The recombinant enzyme (His 6-rAmyKS) was purified in one step using Ni-NTA column affinity with a specific activity of 664.28 U.mg −1. The biochemical properties of the His 6-rAmyKS were determined and compared to those of the non-tagged enzyme. Interestingly, differences were found between the two enzymes mainly for the optimal temperature and pH. Experimental tests and molecular modeling confirmed that the extra residues (C-terminal His-tag fusion peptide and cleavage thrombin site) could be responsible for the slight increase in total activity and the improvement of biochemical properties of the His-tagged enzyme compared to the native one. The His 6-rAmyKS was used as an additive in breadmaking. It showed a significant effect in improving the dough texture and the bread quality.
... SDS-PAGE (under reducing and non-reducing conditions) was carried out with a mini-PROTEAN system (BIO-RAD) using a 4% stacking gel and 12% resolution gel, as described by Laemmli (1970). Samples were dispersed (6 mg protein mL À1 ) in Tris/HCl buffer, pH 8.0 containing 10% (w/v) SDS only (non-reducing buffer) or SDS + 10% (v/v) b-mercaptoethanol (reducing buffer). ...
The gel‐forming ability of the pulse proteins influences the formation of food products with desirable texture, structure, and stability. In this study, Bambara globulin was fractionated into legumin and vicilin for structural and physicochemical analyses, and heat‐induced gel rheological characterisation. Bambara globulin showed major vicilin (7S, Mw: 120 kDa) and minor legumin (11S, Mw: 410 kDa) components by size exclusion chromatography and gel electrophoresis analyses. Legumin had the lowest amount of all the charged amino acids. One basic subunit (22 kDa) was identified in the legumin fraction with predominant helical structures. The sol–gel transition temperatures increased in the order of globulin (40 °C) < legumin (50 °C) < vicilin (80 °C). The G′ and G″ of globulin showed relatively low dependency on heating time beyond the gel point compared to legumin and vicilin subfractions, suggesting a more rapid establishment of its gel network during gelation. Vicilin gel consisted of a microporous structure with a small lath sheet‐like structure compared to others. Bambara vicilin could be a prospective ingredient in the development of new products with defined textural characteristics.
... The sample solution was then centrifuged for 15 min at 12 470 × g (13 000 rpm in a microfuge 16 centrifuge, Beckman Coulter, Inc., Germany) to separate the supernatant and precipitate. Protein analysis of the supernatant was conducted using SDS-PAGE and native PAGE following the method by Laemmli (1970). The bicinchoninic acid test was employed to quantify the concentration of EWP in the supernatant, with BSA serving as the standard (Kralj et al., 2014). ...
Vitamin A (VA) is a fat‐soluble compound that is essential for physiological functions including vision, fetal growth, immune response, cell differentiation and proliferation. However, its susceptibility to environmental conditions leading to poor stability limits its application in food. This study investigated the interaction between egg white protein (EWP) and VA by spectroscopy and molecular docking. The results indicated one EWP could bind 16.00 ± 0.89 VA molecules and the binding constant is (2.59 ± 0.71) × 10⁵ m⁻¹. What's more, there was a fluorescence resonance energy transfer between EWP and VA, and the Förster radius is 5.28 nm. In addition, VA enhanced the hydrophobic interactions with the EWP, resulting in protein aggregation, larger average particle size and more uniform particle size distribution. Finally, the combination of VA with EWP could significantly improve the photothermal stability and storage stability of VA. These studies indicate that EWP nanocomplexes can be used as ideal delivery carriers for bioactive substances.
... SDS-PAGE was performed using 5% loading gel and 10% separation gel [39]. The resulting gel was carefully removed and stained using Coomassie Brilliant Blue R250. ...
Hydrogen peroxide (H2O2) is a chemical that is widely used in many industrial processes, and, except at certain concentrations, it is toxic in biological systems such as water and air. Among enzymes, catalases are important industrial enzymes because of their role in the conversion of hydrogen peroxide to water and molecular oxygen. Herein, catalase (CAT) from Hydnum repandum was purified 3.02-fold with a yield of 68.10% by three-phase partitioning (TPP) for the first time. The purified catalase was immobilised on glutaraldehyde-activated chitosan (Glu-Cts), and its applicability for the removal of hydrogen peroxide released from industrial processes was investigated. The results of the present study showed that the optimum pH and temperature were found to be 7.0 and 30°C for both free and immobilised catalase (CAT-Glu-Cts). The catalytic efficiency (V max/K m) of the immobilised enzyme increased 8-fold compared to the free enzyme. CAT-Glu-Cts was shown to have better pH, thermal stability, and storage stability than free CAT. In this study, >96% of 6 mM, 15 ve 24 mM H2O2 was removed from artificial wastewater after 2 h using immobilised catalase. We expect that CAT-Glu-Cts, obtained by purifying a plant-derived catalase and immobilising it into an environmentally friendly and biocompatible material, is a promising candidate that can be safely used for H2O2 removal in various branches of industry.
... Polyacrylamide gel electrophoresis (PAGE) SDS-PAGE and Native PAGE were both performed on 15% polyacrylamide gels with 6% stacking gels according to Laemmli protocol (Laemmli, 1970 Determination of trypsin and chymotrypsin inhibitor activity on native PAGE gels Native PAGE gels were stained for trypsin and chymotrypsin inhibitor activities as described by Kollipara and Hymowitz (1992). Immediately after separation, NDWI maps corresponded well with NDVI ones according to the stronger photosynthetic activity due to higher canopy water content (Fig. 2). ...
Nowadays, one of the major challenges in the field of agricultural production is an increasing demand for protein in farm animal husbandry. The main source of protein today is still soybean meal, but there is an urgent need to involve alternative protein sources and assess areas of application. Leaf and fruit of black locust (Robinia pseudoacacia L.) could be a possible source of protein, therefore the main composition of different varieties was examined.
Black locust is the most widespread tree species in Hungary occupying approximately 24% of the forested land (465,000 ha) and providing 25% of the annual timber output of the country. The mean wood volume of all black locust forests is 125 m³/ha, with a mean volume of 190 m³/ha at the final cutting age (31 years on average). Black locust foliage could be an important, complementary feed, as it plays the role of rumen filler and during grazing, animals can access other useful plants among the trees. Foliage samples were collected from individuals of black locust cultivars, and subsequent content analyses were carried out. With the help of satellite images, photosynthetic activity and health conditions were investigated in the experimental areas using vegetation and moisture indices on the basis of content values and digestibility studies.
... The peptidoglycan and lipopolysaccharide concentration of the lysates was analyzed by enzyme immunoassay with ELISA kit (Abbexa LCC; Houston, TX, USA), considering the manufacturerʹs instructions. For carbohydrate concentration, the phenol-sulfuric acid colorimetric method [79] was used; for this purpose, the lysates samples were adjusted to 200 µg and 5 µL of 80% phenol and 500 µL of pure H2SO4 were (carefully) added. Samples were incubated for 20 min at 30 °C, followed by spectrophotometer readings at an absorbance of 490 nm [80]. ...
Overuse of antimicrobials has greatly contributed to the increase in the emergence of multidrug-resistant bacteria, a situation that hinders the control and treatment of infectious diseases. This is the case with urinary tract infections (UTIs), which represent a substantial percentage of worldwide public health problems, thus the need to look for alternatives for their control and treatment. Previous studies have shown the usefulness of autologous bacterial lysates as an alternative for the treatment and control of UTIs. However, a limitation is the high cost of producing individual immunogens. At the same time, an important aspect of vaccines is their immunogenic amplitude, which is the reason why they must be constituted of diverse antigenic components. In the case of UTIs, the etiology of the disease is associated with different bacteria, and even Escherichia coli, the main causal agent of the disease, is made up of several antigenic variants. In this work, we present results on the study of a bacterial lysate composed of 10 serotypes of Escherichia coli and by Klebsiella pneumoniae, Klebsiella aerogenes, Enterococcus faecalis, Proteus mirabilis, Citrobacter freundii, and Staphylococcus haemolyticus. The safety of the compound was tested on cells in culture and in an animal model, and its immunogenic capacity by analysing in vitro human and murine macrophages (cell line J774 A1). The results show that the polyvalent lysate did not cause damage to the cells in culture or alterations in the animal model used. The immunostimulatory activity assay showed that it activates the secretion of TNF-α and IL-6 in human macrophages and TNF-α in murine cells. The obtained results suggest that the polyvalent lysate evaluated can be an alternative for the treatment and control of chronic urinary tract infections, which will reduce the use of antimicrobials.
... SDS/PAGE was carried out according to the Laemmli method [50] using 10% polyacrylamide gels. In-gel enzymatic digestion with trypsin was performed according to the previously described procedure [51]. ...
The most extensively studied β‐d‐galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family β‐galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaβGal). Unlike fungal monomeric six‐domain β‐galactosidases, the DaβGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for β‐d‐galactopyranosides, and its distinguishing feature is the ability to cleave pNP‐β‐d‐fucopyranoside. DaβGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °С, and retains activity at 95 °С with a half‐life time value equal to 73 min. These properties make archaeal DaβGal a more attractive candidate for biotechnology than the widely used fungal β‐galactosidases.
... The Laemmli sample buffer (2x) was freshly mixed with a reducing agent at a ratio 1/10 freshly before use. 25 Equal amounts of parasporal protein and sample buffer (1/1) were prepared and boiled for 10 min. The mixture was loaded into wells with 10% TGX Precast Gels (Bio-Rad Laboratories). ...
Historically, the most important source of both antibiotics and anticancer medications has been microorganisms. Bacillus thuringiensis (Bt) is one of the most prominent bacterial species used as a therapeutic agent targeting cancerous cells in recent worldwide investigations. This study was designed to isolate, molecularly identify, and discover novel Saudi Arabian Bt strains that selectively exhibit cytotoxic properties against MDA-MB-231, a human triple-negative breast cancer (TNBC) cell model. The bacterial strain under investigation was biochemically typed using API 20E and API CH50 and molecularly typed using 16S rDNA sequencing. Flow cytometry and immunoblotting were performed to elucidate the mechanism-of-action (MOA). Molecular typing confirmed the identity of the isolated non-hemolytic strain to be Bt and was named Bt HAU-145. Microscopic examination showed that the strain possessed a parasporal (PS) crystal protein with a spherical morphology. Data of cytotoxicity assay based on MTT revealed that Bt HAU-145 strain exhibited selective and potent cytotoxicity against MDA-MB-231, with a 50 percent inhibition (IC50) of 28 µg/ml. FACS analysis revealed that PS proteins induced both late and early apoptosis in a ROS-dependent manner. Immunoblotting assays showed increased expression of caspase-3 in response to PS treatment, paralleled by a reduction in Bcl-2 expression. This is the first study to investigate the MOA of PS proteins from the Saudi Arabian Bt strain, showing an induction of apoptosis through a ROS-dependent mechanism in TNBC cells. It is hoped that PS-based therapeutic strategies will be investigated at the preclinical scale in non-human primates prior to the clinical scale in randomized clinical trials.
... Proteins were analysed by SDS-PAGE in 12.5% polyacrylamide gel according to Laemmli [23]. Proteins were visualized by Coomassie R-250 Brilliant Blue (Sigma-Aldrich, Germany) staining. ...
Background
Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts.
Methods and results
Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph.
Conclusions
The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs’ biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.
... The Laccase purity and molecular mass was visualized with SDS-PAGE using a 12% resolving gel and a 5% stacking gel. At the end of electrophoresis, the gel was stained with Coomassie brilliant blue (Laemmli 1970). The Laccases were purified to homogeneity with a specific activity of 34 U/mg protein. ...
The persistence and ubiquity of polycyclic aromatic hydrocarbons (PAHs) in the environment necessitate effective remediation strategies. Hence, this study investigated the potential of purified Laccases, TlFLU1L and TpFLU12L, from two indigenous fungi Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12), respectively for the oxidation and detoxification of anthracene. Anthracene was degraded with vmax values of 3.51 ± 0.06 mg/L/h and 3.44 ± 0.06 mg/L/h, and Km values of 173.2 ± 0.06 mg/L and 73.3 ± 0.07 mg/L by TlFLU1L and TpFLU12L, respectively. The addition of a mediator compound 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to the reaction system significantly increased the degradation of anthracene, with up to a 2.9-fold increase in vmax value and up to threefold decrease in Km values of TlFLU1L and TpFLU12L. The GC–MS analysis of the metabolites suggests that anthracene degradation follows one new pathway unique to the ABTS system—hydroxylation and carboxylation of C-1 and C-2 position of anthracene to form 3-hydroxy-2-naphthoic acid, before undergoing dioxygenation and side chain removal to form chromone which was later converted into benzoic acid and CO2. This pathway contrasts with the common dioxygenation route observed in the free Laccase system, which is observed in the second degradation pathways. Furthermore, toxicity tests using V. parahaemolyticus and HT-22 cells, respectively, demonstrated the non-toxic nature of Laccase-ABTS-mediated metabolites. Intriguingly, analysis of the expression level of Alzheimer’s related genes in HT-22 cells exposed to degradation products revealed no induction of neurotoxicity unlike untreated cells. These findings propose a paradigm shift for bioremediation by highlighting the Laccase-ABTS system as a promising green technology due to its efficiency with the discovery of a potentially less harmful degradation pathway, and the production of non-toxic metabolites.
... where A 0 and A 10 represent the absorbance of the diluted emulsion immediately after and 10 min after homogenization, respectively, C (g/mL) is the protein concentration before homogenization, and Φ (0.20) is the fraction of the oil volume (v/v) in the emulsion. sulfate (SDS-PAGE) according to the methods of Laemmli [46] using a Mini-Protean Tetra vertical electrophoresis cell (Bio-Rad Laboratories, Inc., USA). The samples were analyzed in gels prepared with 4 and 12 % w/v acrylamide (stacking and separation, respectively) under reducing conditions (with β-mercaptoethanol) and nonreducing conditions (without β-mercaptoethanol). ...
... θ] represents the CD (in millidegrees) obtained from the spectra, n represents the number of amino acid residues (n = 583 for BSA), l is the path length of the cell (1 cm), and C indicates the mole fraction of the protein. For BSA, the helical content is determined from the [θ] value at 208 nm ( ) according to the equation as described by Lu et al.[31]:% helix = {(−[θ]208 − 4 000)/(33 000 − 4 000)} 100(4) ...
... Protein separation was achieved by vertical polyacrylamide gel electrophoresis using sodium dodecyl sulfate under reducing conditions (SDS-PAGE) according to Laemmli (1970) or using a modified method according to Schägger and von Jagow (1987) depending on the mass of the proteins of interest, in an electrophoretic apparatus (SE260, Hoefer). Denaturation of the proteins in the sample was achieved by mixing with sample buffer and subsequent heating (95 °C; 10 min). ...
N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli . We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds.
... SDS PAGE followed Laemmli's methodology [13] to determine the molecular weights of the collagen and gelatin extracted by the different methods. The collagen or gelatin was dissolved in the buffer to obtain a concentration of 1 mg mL −1 . ...
Abattoirs dispose of sheepskins as solid waste due to low price and poor demand for sheepskin leather. In principle, as an alternative to being disposed of in landfill, sheepskins can serve as a source of the protein collagen or the hydrolysis product, gelatin. In this research, sheepskins collected from abattoirs were used as a source of collagen. Three extraction methods were compared: acid extraction, acid with enzymes, and alkali extraction. The extracted material was characterized using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR), small angle X-ray scattering (SAXS), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The collagen and gelatin extraction yield ranged from 3.1% to 4.8% with the product purity determined by hydroxyproline, ranging from 7.8% for the alkali process to 59% and 68% for the acid and acid-enzyme processes. SDS PAGE showed that the acid process produced fragments with molecular weights in the range 100 to >250 kDa, while acid–enzyme resulted in smaller fragments, below 30 kDa. The FTIR region of the amide I band at 1800–1550 cm−1, which was used as an indicator of the collagen and gelatin content, showed that the gelatin dominated in the acid extracts, and the alkaline extract contained a large portion of keratin. SAXS was found to be a sensitive method for showing the presence of intact collagen fibrils in materials from all of the extraction methods, albeit at low concentrations. Herein, sheepskin is shown to be a useful source for collagen–gelatin material of varying molecular weights.
... To determine enzyme production, SDS-PAGE was performed according to the method described by Laemmli (Laemmli 1970). As per this method, a separating gel with an 8% polyacrylamide concentration and a stacking gel with 5% concentration was used. ...
The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.
... A 12% of SDS-PAGE was performed followed the method mentioned by Laemmli applying a small piece of glass plates (8 × 8 cm) gel apparatus (Laemmli, 1970). Molecular weight of crude of enzyme was obtained by using standard molecular weight marker (fermentas). ...
Amylase enzyme is used in various industries due to its diverse applications. In this study, bacteria from soil sample were primarily screened on starch agar medium to identify amylase producer through the detection of prominent clear zone. Total of five soil samples namely bakery points (A-1), sugar cane juice point (A-2), Lichi chinesis garden soil (A-3), rice field (A-4) and sugar industrial waste (A-5) were used in this study. Among the 17 strains isolated from three samples A-1, A-2 and A-3 were found positive for amylase production. The strains were further screened on the production medium. The N-1 bacterial strain revealed higher enzyme activity (92.21 ± 17 IU/ml) compared to the other strain and was thus selected for further work. The strain was identified as Bacillus lichenoformis from the 16S rRNA analysis. Enzyme production was enhanced by optimizing various parameters by one factor at a time technique. The agro industrial waste rice polish was used as substrate. The optimum temperature of the enzyme was 35°C, pH 5.5 and 2% (w/v) of substrate concentration. Qualitative detection by using sodium dodecyl polyacrylamide gel electrophoresis showed that molecular weight of enzyme was 35 kDa. This indicated that the enzyme requires a moderately high temperature and neutral pH to show greatest activity.
... Protein concentrations were measured using Bio-Rad protein assay kit (Bio-Rad Catalog Number 500-0006). Proteins were separated on 10% polyacrylamide gels as described previously (Laemmli 1970). Both anti-his-tag antibody (Pierce, Cat. ...
CIA5 is a zinc-finger containing transcription regulator reported to be a master regulator of the critically important, inducible CO 2 -concentrating mechanism of the model, unicellular green alga, Chlamydomonas. Although mutants in the CIA5 gene facilitated identification of CIA5 more than two decades ago, we still know little about the detailed function of this important protein. Here we report the first successful over-expression of full length CIA5 proteins in E. coli , confirmed by SDS-PAGE and Western immunoblots. We also used these purified, full length CIA5 proteins to identify potential specific DNA-binding sequences using random binding site selection (RBSS), which was confirmed using a gel mobility shift assay (GMSA) to demonstrate highly specific protein-DNA interaction with purified, full-length CIA5. In addition, we identified a 9-bp GC rich (GGGGCGGGG) motif from the promoters of CIA5 dependent genes, and demonstrated using GMSA that promoter fragments containing this candidate motif from three CIA5-dependent genes also showed highly specific protein-DNA interaction with CIA5, although the GMSA interactions were somewhat weaker than with the RBSS-identified sequence. Nonetheless, this work clearly provides the first direct evidence that CIA5 can bind specific DNA sequences in vitro and thus opens the way for more extensive in vivo experiments to determine whether the specific DNA-binding of CIA5 has any biological relevance in vivo .
... Protein denaturation was performed at 90 • C for 5 min. The same amounts (15 µg) of protein extracts were loaded and separated on 10% SDS-PAGE (1 mm polyacrylamide gel) according to the procedure described in [70]. After the proteins were separated, they were blotted onto low-fluorescence PVDF membranes (0.45 µm, Bio-Rad Laboratories, Inc., Hercules, CA, USA) (30 min, 25V/1A) using a semi-dry transfer (Bio-Rad Trans-Blot Turbo Transfer System, Bio-Rad Laboratories, Inc., Hercules, CA, USA). ...
Winter plants acclimate to frost mainly during the autumn months, through the process of cold acclimation. Global climate change is causing changes in weather patterns such as the occurrence of warmer periods during late autumn or in winter. An increase in temperature after cold acclimation can decrease frost tolerance, which is particularly dangerous for winter crops. The aim of this study was to investigate the role of brassinosteroids (BRs) and BR analogues as protective agents against the negative results of deacclimation. Plants were cold-acclimated (3 weeks, 4 °C) and deacclimated (1 week, 16/9 °C d/n). Deacclimation generally reversed the cold-induced changes in the level of the putative brassinosteroid receptor protein (BRI1), the expression of BR-induced COR, and the expression of SERK1, which is involved in BR signal transduction. The deacclimation-induced decrease in frost tolerance in oilseed rape could to some extent be limited by applying steroid regulators. The deacclimation in plants could be detected using non-invasive measurements such as leaf reflectance, chlorophyll a fluorescence, and gas exchange monitoring.
... Cells were lysed in hot Laemmli sample buffer (Laemmli 1970). Protein concentration was determined according to (Smith et al. 1985). ...
Tert-butyl hydroperoxide (t-BuOOH) is an organic hydroperoxide widely used as a model compound to induce oxidative stress. It leads to a plethora of cellular damage, including lipid peroxidation, DNA double-strand breaks (DNA DSBs), and breakdown of the mitochondrial membrane potential (MMP). We could show in several cell lines that t-BuOOH induces ferroptosis, triggered by iron-dependent lipid peroxidation. We have further revealed that not only t-BuOOH-mediated ferroptosis, but also DNA DSBs and loss of MMP are prevented by cell–cell contacts. The underlying mechanisms are not known. Here, we show in murine fibroblasts and a human colon carcinoma cell line that t-BuOOH (50 or 100 µM, resp.) causes an increase in intracellular Ca²⁺, and that this increase is key to lipid peroxidation and ferroptosis, DNA DSB formation and dissipation of the MMP. We further demonstrate that cell–cell contacts prevent t-BuOOH-mediated raise in intracellular Ca²⁺. Hence, we provide novel insights into the mechanism of t-BuOOH-triggered cellular damage including ferroptosis and propose a model in which cell–cell contacts control intracellular Ca²⁺ levels to prevent lipid peroxidation, DNA DSB-formation and loss of MMP. Since Ca²⁺ is a central player of toxicity in response to oxidative stress and is involved in various cell death pathways, our observations suggest a broad protective function of cell–cell contacts against a variety of exogenous toxicants.
... The protein content was determined by Bradford assay (Bradford, 1976). Forty micrograms of proteins in the lysates were adjusted to contain 2% SDS (30 mM Tris-HCl pH 6.8, 10% glycerol, 0.01% bromophenol blue, and 5 mM dithiothreitol, DTT), boiled for 5 min and loaded in SDS-polyacrylamide gels (Laemmli, 1970). After electrophoresis, the proteins were transferred to 0.45 μm nitrocellulose in a semidry Biorad apparatus at 25 V for 40 min. ...
Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas disease. The parasite has developed various mechanisms to get through its intricate life cycle and adapt to different evolutionary phases. T. cruzi proliferates in the insect vector's digestive tract as an epimastigote form, encountering fluctuating nutrient availability and oxidative stress caused by the digestion of red blood cells from the mammalian host blood meal. To unravel how the parasite's metabolism adapts to these changing conditions, we conducted an analysis of the chemical species present in epimastigote forms. This involved comparing cultured parasites with those subjected to nutritional deficiency or oxidative stress using untargeted metabolomics. We looked at 21 samples: seven biological copies of parasites that were actively growing, seven samples that were put in a medium without nutrients for 3 h, and seven samples that were treated with glucose oxidase for 30 min to make H2O2 continuously. Importantly, in all conditions, parasite viability was maintained when the samples were collected. Upon nutrient removal, we observed a substantial decrease in amino acids and carbohydrate metabolites, accompanied by the accumulation of fatty acids and steroids, with the predominance of inositol and sphingolipid metabolism, along with a simultaneous decrease in the levels of H2O2. In the presence of H2O2, a significant rise in components of the pentose pathway and specific amino acids such as methionine and serine occurred, along with pathways related to an increase in antioxidant species metabolism such as ribulose 5‐phosphate and glyceric acid. Conversely, fatty acid and steroid levels decrease. We found no common increase in metabolites or lipids. In contrast, eight species (succinic acid, glutamic acid, valine, 2‐hydroxyisocaproic acid, alanine, indolelactic acid, proline, and lanosterol) were consumed under both stresses. These findings underscore the rapid and distinct enrichment responses in amino acids, lipids, and carbohydrates required to cope with each different environmental condition. We concluded that T. cruzi presents a flexible metabolism that rapidly adapts to variable changes in the environment.
... Following the addition of trypsin, 9 µl aliquots were taken from the reaction at specific time intervals and added to the SDS-PAGE loading buffer to stop the reaction. Then, 3 µg of protein was loaded onto an 8-20% homemade polyacrylamide gradient gel, and the digestion products were separated according to Laemmli [45]. To assess the effect of the long incubation time on the digestion products, additional gN1 samples were prepared. ...
Background
Nucleobindin-2 (Nucb2) and nesfatin-1 (N1) are widely distributed hormones that regulate numerous physiological processes, from energy homeostasis to carcinogenesis. However, the role of nesfatin-2 (N2), the second product of Nucb2 proteolytic processing, remains elusive. To elucidate the relationship between the structure and function of nesfatins, we investigated the properties of chicken and human homologs of N1, as well as a fragment of Nucb2 consisting of N1 and N2 conjoined in a head-to-tail manner (N1/2).
Results
Our findings indicate that Zn(II) sensing, in the case of N1, is conserved between chicken and human species. However, the data presented here reveal significant differences in the molecular features of the analyzed peptides, particularly in the presence of Zn(II). We demonstrated that Zn(II) has a Janus effect on the M30 region (a crucial anorexigenic core) of N1 and N1/2. In N1 homologs, Zn(II) binding results in the concealment of the M30 region driven by a disorder-to-order transition and adoption of the amyloid fold. In contrast, in N1/2 molecules, Zn(II) binding causes the exposure of the M30 region and its destabilization, resulting in strong exposure of the region recognized by prohormone convertases within the N1/2 molecule.
Conclusions
In conclusion, we found that Zn(II) binding is conserved between chicken and human N1. However, despite the high homology of chicken and human N1, their interaction modes with Zn(II) appear to differ. Furthermore, Zn(II) binding might be essential for regulating the function of nesfatins by spatiotemporally hindering the N1 anorexigenic M30 core and concomitantly facilitating N1 release from Nucb2.
... The TMP was fixed at 9.72 kPa, and, for the diafiltration step, 8 volumes were used with both membranes, followed by a final concentration step using a 100-30 kDa cut-off membrane. The results obtained from the diafiltration and concentration processes were analyzed by electrophoresis according to Laemmli [18]. The fouling of the membrane during the protein concentration stage was studied according to the mathematical models of Hermia [19]. ...
Inflammatory bowel disease (IBD) is an autoimmune disorder caused by uncontrolled immune activation and the subsequent destruction of the colon tissue. Quercetin (Qt) is a natural antioxidant and anti-inflammatory agent proposed as an alternative to mitigate IBD. However, its use is limited by its low oral bioavailability. This study aimed to develop nanoemulsions (NEs) based on a soluble chenopodin/alginate (QPA) complex and Tween 80 (T80), intended for the colonic release of Qt, activated by the pH (5.4) and bacteria present in the human colonic microbiota. NEs with different ratios of QPA/Tw80 (F1-F6) were prepared, where F4Qt (60/40) and F5Qt (70/30) showed sizes smaller than 260 nm, PDI < 0.27, and high encapsulation efficiency (>85%). The stability was evaluated under different conditions (time, temperature, pH, and NaCl). The DSC and FTIR analyses indicated hydrophobic and hydrogen bonding interactions between QPA and Qt. F4Qt and F5Qt showed the greater release of Qt in PBS1X and Krebs buffer at pH 5.4 (diseased condition), compared to the release at pH 7.4 (healthy condition) at 8 h of study. In the presence of E. coli and B. thetaiotaomicron, they triggered the more significant release of Qt (ƒ2 < 50) compared to the control (without bacteria). The NEs (without Qt) did not show cytotoxicity in HT-29 cells (cell viability > 80%) and increased the antioxidant capacity of encapsulated Qt. Therefore, these NEs are promising nanocarriers for the delivery of flavonoids to the colon to treat IBD.
... The molecular weight of the lipase was determined via SDS-PAGE analysis following the method of Laemmli (1970) using standard protein markers: α-Lactalbumin (14.3 kDa), Trypsin inhibitor (20.1 kDa), Carbonic anhydrase (29 kDa), IgG (50 kDa), Bovine serum albumin (66 kDa), and Phosphorylase B (97.4 kDa). The SDS-PAGE analysis (Bio-Rad, CA) was conducted with a resolving gel of 12% acrylamide and a stacking gel of 4.5% acrylamide. ...
The lipase enzyme was isolated and purified from Staphylococcus argenteus MG2 (MTCC 12820) to homogeneity using ammonium sulphate precipitation followed by chromatographic techniques. This process resulted in a purification factor of 40.96-fold and a 26.25% recovery with a specific activity of 744.68 U mg-1. The molecular weight of the purified lipase was determined by SDS-PAGE to be 45 kDa. The Km and Vmax values of the purified lipase were calculated to be 4.95 mM and 79.36 µmol/min/mg-1, respectively. The maximum lipase activity was observed at pH 7.0 and 30 ºC with 100% stability, and it was also found to be stable in a broad range of pH (5-12) and temperature (30-90 ºC). The enzymatic activity of this Staphylococcal lipase was increased by Ca2+ to 105.71% at a concentration of 1 mM CaCl2. Additionally, it exhibited marked stability and activity in organic solvents. In the presence of 1% SDS surfactant, it retained 85.16% residual activity, while the metal chelator EDTA (inhibitor) reduced the lipase activity to 83.87% residual activity at a concentration of 1% w/v. This alkali-stable and thermo-stable lipase can be exploited by extending its use in the preparation of detergents and in various industrial and biotechnological applications.
... The samples protein concentration was determined by BCA assay (Pierce Thermo Scientific, Rockford, IL, USA). Protein samples were analysed using 10-15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [25]. SDS-PAGE gels were stained with Coomassie blue and scanned (iBright 1500, Invitrogen, Waltham, MA, USA). ...
A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.
... SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to analyze the protein composition of the various surimi samples based on the method originally reported by Laemmli et al. [41] and Zhang et al. [42] with some modifications. The samples from the BBD experiments were weighed into 0.1 g pieces and placed in 15 mL centrifuge tubes with 2 mL of the sample buffer. ...
Silver Carp (SC) is an under-utilized, invasive species in North American river systems. In this study, the synergistic effects of manufactured Microfiber (MMF), Transglutaminase (TG), and chicken skin collagen (CLG)) to enhance surimi gel quality from frozen SC were studied. The gel strength, textural properties, rheological properties, water-holding capacity (WHC), water mobility, microstructure, and protein composition of the gel samples were determined to assess the impact of the additives individually and synergistically. The results suggested that TG had the most pronounced effect on the surimi gel properties by promoting protein cross-linking. Synergistic effects between TG, MMF, and CLG can bring effective gel property enhancement larger than the individual effect of each additive alone. With the established response-surface models, the combination of CLG and MMF can be optimized to produce surimi gels with less TG but comparable in properties to that of the optimal result with high TG usage. The findings of this study provided a technical foundation for making high-quality surimi gel products out of frozen-stored SC with synergistic utilization of additives, which could serve as guidelines for the industrial development of new surimi products.
... Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Protein profiles of select samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions according to Laemmli,21 with modifications. Samples were solubilized in 4× LDS buffer (lithiumdodecyl sulfate buffer; Genscript, Piscataway, NJ, USA) at room temperature for 2 h, then heated in a 70°C water bath for 10 min, and 250 mL L −1 ⊎-mercaptoethanol was added to the samples under reducing condtions. ...
BACKGROUND
High moisture meat analog (HMMA) products processed using extrusion have become increasingly popular in the last few years. Because the formation of disulfide bonds is believed to play a critical role in the texturization mechanism, this study aimed to understand how chemical compounds capable of reducing disulfide bonds, specifically cysteine, sodium metabisulfite, and glutathione, affect the texture and the chemical interactions between the proteins.
METHOD
Wheat protein blended with cysteine, sodium metabisulfite, or glutathione at levels of 0, 0.5, 1.0, 2.5, 5.0, and 7.5 g kg⁻¹ was extruded at three different temperatures (115, 140, and 165 °C) using a co‐rotating twin‐screw extruder. The feed rate (85 g min⁻¹), the moisture content (600 g kg⁻¹), and the screw speed (300 rpm) were kept constant. Unextruded and extruded material was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, polymeric protein fractionation, and sulfhydryl group/disulfide bond analysis. Extruded samples were further analyzed for their hardness and their anisotropic index.
RESULTS
The inclusion of reductants significantly affected the structure of the obtained extrudates. Although reducing agents had a relatively small impact on the total amount of disulfide bonds, their action significantly enhanced crosslinking between the proteins. At select conditions, samples with high fibrousness were specifically obtained when cysteine or sodium metabisulfite was included at levels of 5.0 g kg⁻¹.
DISCUSSION
In the presence of reducing agents, it is believed that disulfide bonds are split earlier during the process without binding to them, giving the protein strands more time to unravel and align, leading to a better flow behavior and more fibrous products. © 2024 Society of Chemical Industry.
... For total protein extracts, 5 × 10 7 T. cruzi cells (log-phase epimastigotes, culture-derived tripomastigotes or axenic amastigotes) were harvested by centrifugation at 1200g for 10 min. After washing, cells were directly lysed in Laemmli sample buffer [50]. interactions (dashed orange lines) are shown. ...
Trypanosoma cruzi is the causative agent of Chagas disease, as well as a trypanosomatid parasite with a complex biological cycle that requires precise mechanisms for regulating gene expression. In Trypanosomatidae, gene regulation occurs mainly at the mRNA level through the recognition of cis elements by RNA-binding proteins (RBPs). Alba family members are ubiquitous DNA/RNA-binding proteins with representatives in trypanosomatid parasites functionally related to gene expression regulation. Although T. cruzi possesses two groups of Alba proteins (Alba1/2 and Alba30/40), their functional role remains poorly understood. Thus, herein, a characterization of T. cruzi Alba (TcAlba) proteins was undertaken. Physicochemical, structural, and phylogenetic analysis of TcAlba showed features compatible with RBPs, such as hydrophilicity, RBP domains/motifs, and evolutionary conservation of the Alba-domain, mainly regarding other trypanosomatid Alba. However, in silico RNA interaction analysis of T. cruzi Alba proteins showed that TcAlba30/40 proteins, but not TcAlba1/2, would directly interact with the assayed RNA molecules, suggesting that these two groups of TcAlba proteins have different targets. Given the marked differences existing between both T. cruzi Alba groups (TcAlba1/2 and TcAlba30/40), regarding sequence divergence, RNA binding potential, and life-cycle expression patterns, we suggest that they would be involved in different biological processes.
... The final protein preparations were stored frozen in the elution buffer used at the gel filtration step (0.1 M Mops/KOH, pH 7.2, 2 mM MgCl2, 0.1 mM CoCl2, and 150 mM KCl). The purity of the isolated proteins, as estimated by SDS-PAGE [34] with Coomassie staining, was >90%. Protein concentrations in milligrams per milliliter were determined spectrophotometrically using the extinction coefficient A280 0.1% calculated from the amino acid composition with ProtParam (https://web.expasy.org/protparam/ ...
Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the “half-of-the-sites” ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a “two non-interacting pairs of interacting regulatory sites” concept in CBS-PPase regulation.
In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg⁻¹. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg⁻¹) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg⁻¹). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5ʹ-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme’s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL⁻¹ and 1580.3 ± 183.7 μmol mg⁻¹ protein min⁻¹, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.
Melatonin (Mel) is a phytohormone that plays a crucial role in various plant processes, including stress response. Despite numerous studies on the role of Mel in stress resistance, its significance in plants exposed to benzalkonium chloride (BAC) pollution remains unexplored. BAC, a common antiseptic, poses a threat to terrestrial plants due to its widespread use and inefficient removal, leading to elevated concentrations in the environment. This study investigated the impact of BAC (0.5 mg L-1) pollution on wild-type Col-0 and snat2 knockout mutant Arabidopsis lines, revealing reduced growth, altered water relations, and gas exchange parameters. On the other hand, exogenous Mel (100 μM) treatments mitigated BAC-induced phytotoxicity and increased the growth rate by 1.8-fold in Col-0 and 2-fold in snat2 plants. snat2 mutant seedlings had a suppressed carbon assimilation rate (A) under normal conditions, but BAC contamination led to further A repression by 71% and 48% in Col-0 and snat2 leaves, respectively. However, Mel treatment on stressed plants was successful in improving Fv/Fm and increased the total photosynthesis efficiency by regulating photochemical reactions. Excessive H2O2 accumulation in the guard cells of plants exposed to BAC pollution was detected by confocal microscopy. Mel treatments triggered almost all antioxidant enzyme activities (except POX) in both Arabidopsis lines under stress. This enhanced antioxidant activity, facilitated by foliar Mel application, contributed to the alleviation of oxidative damage, regulation of photosynthesis reactions, and promotion of plant growth in Arabidopsis. In addition to corroborating results observed in many agricultural plants regarding the development of tolerance to environmental stresses, this study provides novel insights into the action mechanisms of Mel under the emerging pollutant benzalkonium chloride.
The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5' end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania .
Lectin (TfgL) was purified from the seeds of Trigonella foenum-graecum (Fenugreek) belonging to fabaceae family by ammonium sulphate precipitation, ion exchange and followed by size exclusion chromatography. SDS–PAGE analysis revealed that TfgL molecular weight is approximately 27 kDa. 2D-PAGE reveals the existence of two isolectins (pI values of 6.3 and 6.7) with acidic nature and charge heterogeneity. The MALDI-TOF–MS and peptide mass fingerprinting investigation of TfgL showed sequence similarity with a lectin. The hemagglutinating activity of TfgL was stable in broad range of temperature 37–90 °C and at varied pHs 3, 7.6 and 10. Far-UV circular dichroism measurements showed that TfgL is mostly composed of α-helix (84.5%), β-sheet (6.5%), β-turns (5%) and unordered structures (4%). TfgL showed conformational stability in wide range of temperatures (20‒90 °C) and pHs (3, 7.6 and 10) but lost its secondary structure in the presence of 6 M Gdn.HCl. Quenching titrations were carried out with acrylamide and iodide quenchers in order to investigate the exposure and accessibility of the protein tryptophan residues. Maximum quenching observed with acrylamide compared to iodide revealed that the Trp residues of TfgL are buried in the protein core, which is hydrophobic in nature. TfgL showed binding affinity towards porphyrin, the association constant (Ka), for MnTSPP and MnTMPyP was calculated to be 1.2 × 106 M‒1 and 3.45 × 106 M‒1, respectively.
The product of gene 31 (P31) of bacteriophage T4 is required for the formation of the phage capsid and its related structures. In the absence of active P31, product P23, the major component of the phage capsid, aggregates into “lumps” which sediment with the cell envelope. Temperature-shift experiments with ts-mutants in gene 31 demonstrate that the P23 aggregates can be dissolved by activated P31 and the dissolved P23 is normal, in that it can be used for incorporation into active phage. It is possible that P31 acts catalytically.Two different ts-mutants in gene 31 produce two different temperature-sensitive proteins. One is irreversibly inactivated if produced at the restrictive temperature; but when synthesized at the permissive temperature, it becomes heat stable and remains functional at the restrictive temperature. The other is reversibly affected by temperature, activated following shift to permissive temperature and inactivated if restrictive temperature is established.
Two series of experiments were performed, utilizing a modification of the hemolysin plaque technique which registers 19S antibody, in an attempt to determine the frequency of cells capable of simultaneously producing antibody to two non-cross-reacting antigens. Mice were immunized i.v. with rabbit and camel RBC and their spleens assayed for cells producing antibody against both antigens. 16,904 cells producing antibody of one or the other specificity, from 26 mice, were counted. Not one cell was detected which produced antibody of two specificities. Rabbits were immunized intradermally with HSA to which polyalanyl and p-azobenzenearsonate groups were chemically attached. The individual haptens, polyalanyl, and p-azobenzenearsonate groups were coupled to separate aliquots of SRBC, and the lymph nodes of immunized rabbits were assayed for cells releasing antibody against both haptens. In a study of 11 rabbits, after counting 27,845 cells producing antibody, we detected no "double" plaques.
Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Viñuela, and Maizel (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.
Excerpt
INTRODUCTION
Following infection of a sensitive bacterium with a phage, a characteristic series of intracellular events occur. In the case of the virulent phage T4, these events include both the cessation of synthesis of many macromolecular constituents characteristic of the growing bacterial cell, and the establishment of a new biosynthetic pattern directed toward the growth and reproduction of the phage. In this new pattern of events, one set of synthetic activities follows another in temporal sequence. For example, a series of enzymes concerned with the synthesis of phage-specific DNA are formed during the first ten minutes following infection while the protein components of the phage particles are synthesized later (see, for example, Kellenberger, 1961). These events are due to the introduction of the phage genome into the bacterial cell and it becomes, therefore, of basic interest to understand how the phage genome is implicated in these processes. This problem which...
Two beginning sequences, pppGpGpGpGpApApCp ... and pppGpGpGpGpGpApApCp ..., have been identified in the RNA of bacteriophage Qbeta. Both were consistently found in nearly equal amounts
The formation of bacteriophage T4 has been studied by characterizing the phage components accumulating in cells infected—under restrictive conditions—with mutants blocked at different stages of the assembly process. Three structures which appear to be intermediates in tail assembly have been isolated by centrifugation: baseplates (S20,w ≅ 80 s), core-baseplates (S20,w ≅ 80 s), and core-baseplates with surrounding sheath (S20,w ≅ 130 s). The functions of genes 19, 48 and 54 are required for the conversion of the baseplate to the core-baseplate. The functions of genes 3, 15 and 18 are required for the formation of the sheath. Gene 18 codes for a major structural protein of the sheath. Genes 3 and 15 specify products required for the stabilization and completion of the sheath.Tail assembly is sequenced; the baseplate is completed first and the core forms on the baseplate. The gene 18 product polymerizes on the core-baseplate and then the 3 and 15 gene products fix the sheath subunits in the polymerized form. After sheath formation, a segment appears to be added to the core at the terminus of the sheath, permitting subsequent attachment of the head. The tail fibers attach only after the particle formed by head-tail union has been acted upon by the gene 9 product. Particles which have not been acted upon by the gene 11 or 12 product adsorb to bacteria but do not kill them.Electron microscopic observations on the state of phage heads in mutant lysates are also presented. Mutations in three genes result in the accumulation of head membranes empty of DNA.Phage heads and phage tails are formed independently of each other.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
Conditional lethal mutations in gene 20 lead to the production of polyheads. Observed intracellularly in sections, polyheads are tubular structures. The cross sections are frequently pentagonal. Polyheads are filled with an unidentified internal substance which is different—at least in concentration—from the contents of normal phage. In a lysate most polyheads are open, but a few are sealed at one or both ends; most open polyheads appear empty upon examination. Serological studies show that polyheads and normal heads have at least one antigenic site in common. The normal heads have at least one more antigenic site than the polyheads.To produce polyheads, the normal functions of two “head” genes are necessary: (a) gene 23 which is believed to produce the protein subunit of the capsid, and (b) gene 31, the function of which has not yet been identified.A temperature-sensitive mutation L65 in gene 23 produces abnormal phage heads. This abnormality is also observed in polyheads produced by the double mutant tsL65 (in gene 23) -amN50 (in gene 20).
When particles of phage T2 are subjected to osmotic shock and the resulting solution is digested with deoxyribonuclease and centrifuged for 5 hours at 93,000g, the supernatant fraction contains 7% of the acid-insoluble sulfur (or about 7% of the total protein) of the particles. The protein fraction (“internal protein”) separated in this way is virtually free from phage coat protein, contains all the internal antigen of the particles, and small amounts of at least one other protein.In solutions prepared by submitting phage particles to osmotic shock but without enzymatic treatment, the internal protein associates reversibly with the nucleic acid at low salt concentrations. It dissociates almost completely at NaCl concentrations greater than 0.1M, at MgCl2 concentrations greater than 0.01M, or at pH greater than 10.In infected bacteria, protein accumulates that is a precursor of the internal protein of phage particles and can be assayed as such by radiochemical methods. Measured in this way, synthesis of internal protein begins promptly after infection, reaches a maximum rate after about 10 minutes, and falls to a somewhat lower rate thereafter. The rate of synthesis is about equal to the rate of incorporation into phage particles after these start to form. The precursor pools contain about 54 phage-equivalent units of internal protein, as compared to 28 units of phage coat protein and 45 units of nucleic acid.Synthesis of internal protein is stopped by addition of chloramphenicol to the culture under conditions that permit continued synthesis of nucleic acid. The rate of incorporation of labeled, early-synthesized internal protein into phage particles is independent of the amount of phage-precursor nucleic acid in the cells when the latter is varied by appropriate chloramphenicol treatment. This is true even when the amount of precursor nucleic acid exceeds the equivalent amount of precursor internal protein, and it shows that if internal protein makes any persistent attachment to phage nucleic acid inside bacteria, it does so at the time of maturation of phage particles, not at the time of synthesis of nucleic acid.Labeled internal protein of the infecting phage particles does not reappear as internal protein in the offspring phage particles.
The protein product of the A cistron (the maturation protein) of bacteriophage R17 has been identified as a structural component of the virus particle. Examination of wild-type R17 labeled with histidine (an amino acid not present in the coat protein of the phage) reveals the existence of one major polypeptide which co-electrophoreses with the A cistron product extracted from actimomycin-treated infected spheroplasts. The A protein, moreover, is totally absent from the uninfective defective particles produced by the growth of three A cistron amber mutants in non-permissive hosts. Gel filtration on Sephadex G200 yields a molecular weight estimate of 35,000 to 40,000 for the histidine-labeled A protein monomer. It can be present in no more than several copies, since on the average only four molecules of histidine are associated with each wild-type phage particle.
A high-frequency transducing element for erythromycin resistance in Staphylococcus aureus has been found. This element, P11de, is apparently the result of a recombinational event between a temperate phage, P11, and a penicillinase plasmid, γ; it lacks substantial sections both of the plasmid and of the bacteriophage. The phage moiety is cryptic, conferring neither lysogeny nor superinfection immunity upon host cells carrying it; helper phage is required for the production of transducing particles but not for the transduction process. Demonstrable phage-related properties include reactivation of ultraviolet-inactivated homologous phage and rescue of temperature-sensitive phage mutants.The plasmic moiety includes an erythromycin resistance marker, and the mc region, which is responsible for plasmid compatibility, is involved in a specific host-plasmid interaction and is required for plasmid replication; all the other known plasmid markers are missing. The autonomous replication of P11de as well as its intracellular behavior vis-à-vis prophages and other plasmids appear to be governed by its mc determinant. In these respects P11de is similar to the parental γ plasmid. This plasmid control, however, is superseded by active P11, which induces P11de to multiply extensively.P11de can recombine with wild-type plasmids; the resulting recombinants are not transduced at high frequency, nor do they reactivate UV inactivated phage particles. Based on these findings, a possible genetic structure for P11de is presented and compared with maps of naturally occurring plasmids.
Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium → glutamine → glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium → glutamate → glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of α-ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.
The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N2-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH3-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K
m
for ammonium predominates in the N2-grown cell.
Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.
Studies were performed with tryptophan-operon polarity mutants to determine whether normal numbers of tryp-mRNA molecules were produced by these strains and whether the short tryp-mRNA molecules detected (Imamoto & Yanofsky, 1967) resulted from selective degradation of regions of the intact tryp-mRNA. In experiments in which pulse-labeling was performed immediately after the shift of bacterial cultures from repression to derepression conditions, normal production of early tryp-mRNA was observed in all polarity mutants examined. When pulse-labeling was performed at different times prior to the appearance of the first intact tryp-mRNA molecules in the wild-type strain (before six minutes after the initiation of derepression), reduced levels of tryp-mRNA were detected in the polar mutants and the mRNA region corresponding to the operon region beyond the nonsense codon was conspicuously absent. Since the growing mRNA chain is presumably attached at the site of synthesis during this period, it seems unlikely that selective degradation of intact tryp-mRNA molecules could be responsible for the short mRNA molecules that are detected. It was found that decreasing the pulse period in transcription experiments with polarity mutants failed to increase the relative amount of total detectable tryp-mRNA or the relative level of the tryp-mRNA regions corresponding to the genes of the operon beyond the gene with the polarity mutation. In addition, sucrose gradient sedimentation studies failed to detect a shift of the tryp-mRNA profile of polarity mutants to higher molecular weight regions as the pulse time was decreased. These findings suggest that polarity mutations may cause premature termination of transcription of an operon in the vicinity of an introduced nonsense codon.
Proteins made by E. coli cells infected with bacteriophage T4 were analyzed by a method which combined disc electrophoresis and autoradiography. The precursorproduct relationship between subunits and larger components (head, tail) was studied by taking advantage of the fact that the larger components cannot penetrate into the gel used in disc electrophoresis.These studies have shown that the early proteins are not controlled as a single homogeneous class all members of which are synthesized during the same time periods; instead, they start being formed and are shut off at various times during the early part of the infection process.Amber mutants in gene 30 (polynucleotide ligase-defective) synthesized a small amount of DNA, which was later degraded to a fraction soluble in trichloroacetic acid. The ligase-defective mutants were capable of synthesizing an almost normal amount of late proteins, whereas all other DNA-negative mutants, including a deoxycytidine triphosphatase(dCTPase)-defective mutant and maturation-defective mutants could not induce late protein synthesis. The dCTPase-defective mutant, which also synthesizes a small amount of unstable DNA, did not induce late protein synthesis even when the degradation of DNA was prevented.
By applying analytical acrylamide electrophoresis to degraded purified capsid-related material, we found that (1) normal T4 capsids contain a major component, identified as a product of gene 23, plus at least two minor components, k and l, which we could not yet identify genetically; (2) capsids of the short-headed variant contain the same components as the normal ones; and (3) polyheads contain mainly the product of gene 23 and very little, if any, of the minor components k and l.The minor components can be extracted from capsids by treatment with 8 M urea at 45°. A residual capsid is left behind which, in the electron microscope, is not significantly different from the normal one.It is discussed why k and l are not likely to have a morphopoietic role, since they are identifiable neither with the product of genes 66 and 20, whose morphopoietic functions are known, nor with that of other genes known to be necessary for the production of stable heads.
The subunits of T2 head protein prepared by alkaline or acetic acid degradation form four bands in acrylamide gel electrophoresis. The major band M was confirmed as the product of gene 23. The significance of the k, l, and g bands is considered. T2 and T4 head protein preparations differ in the k band, but not in the M band. The method was not sufficiently sensitive to distinguish the head protein of T4 from that of the amber mutant in gene 23 grown in the permissive cell CR63; nor did it distinguish T2 head protein from the various ht mutants tested. The preparation of stable subunits is discussed.
Capsids of bacteriophage T4 have been dissociated in three different solvents: (1) 6 M guanidine hydrochloride, (2) 6 M guanidine hydrochloride plus 0.1 M β-mercaptoethanol, and (3) 67% acetic acid. Sedimentation equilibrium experiments done in the presence of mercaptoethanol have shown that the capsid subunits can be divided into two classes with molecular weights of approximately 11,000 and 46,000. In the absence of mercaptoethanol, a component of molecular weight 78,000 has been found, indicating that disulfide bonds may be important in stabilizing the phage head structure.
The effect of increasing concentrations of urea on sedimentation velocity, enzymatic activity, antigenicity, and protein fluorescence of ß-galactosidase (ß-d-galactoside galactohydrolase, EC 3.2.1.23) indicates that the enzymatically active tetramer is completely dissociated into inactive monomer in 6 M urea. Removal of urea by dialysis results in reaggregation to the active tetramer together with a return to normal values of those properties which have been studied. Protein which is immunologically related to ß-galactosidase but incapable of forming an enzymatically active tetramer was added to wild-type enzyme in the presence of 8 M urea. After renaturation, the enzymatically active tetramer was found to be a hybrid consisting of mutant and wild-type subunits containing full enzymatic activity.
The complex structure of bacteriophage T4 includes a variety of proteins which become assembled into mature particles during intracellular development of the virus. Some insight into the genetic control of this process has been provided by physiological studies with conditional lethal mutants, which show that over 40 phage genes are involved in T4 morphogenesis (Fig. 1). However, the mechanisms by which components are assembled have remained obscure, due in part to the lack of a suitable system for their study. In the experiments reported below, conditional
lethal mutants of strain T4D have been exploited to develop an in vitro system in which several of the steps in phage morphogenesis can be demonstrated.
Ornstein and Davis (1964) have introduced a method, called “disc electrophoresis”, which yields extremely high resolution of proteins electrophoresed in cylindrical columns of polyacrylamide gel. By means of disc electrophoresis, a large number of protein components in a complex mixture can be separated and detected in a single operation. Bands of C14-labeled proteins in disc electropherograms can be detected using techniques described by Heideman (1964); and Jovin, Chrambach, and Naughton (1964). This report presents an alternative procedure involving autoradiography of dried longitudinal gel slices. The method is relatively uncomplicated and can be used to develop the entire pattern of radioactivity in a gel without significant sacrifice of resolution.
Three acid-soluble components have been detected in E. coli infected with bacteriophage T4D. These components first appear in infected cells at 11 minutes after infection (at 37 °), and two of them are incorporated into mature phage particles from which they can be released by osmotic shock as well as by trichloroacetic acid extraction. The properties of these components indicate that they are polypeptides of several thousand molecular weight. They thus appear to correspond, at least in part, to the acid-soluble peptide fraction in T2H phage particles described earlier by Hershey (1957).Conditionally lethal (amber) mutants of T4D blocked in head formation fail to produce all three of the components in a nonpermissive host. This is true for mutants affected in any of six different genes. By contrast, mutants blocked in other kinds of assembly functions are able to produce the components. This finding suggests that the appearance of the components is associated with formation of the phage head.A delay of several minutes between the incorporation of a labeled amino acid into protein and the appearance of label in the acid-soluble components indicates that that these components arise from a precursor.These findings suggest that during phage maturation a protein is encapsulated with the phage DNA and is subsequently fragmented to yield the acid-soluble components which remain trapped inside the phage head. Some possibilities are discussed concerning the relation of this process to phage maturation.
The physiological manifestation of conditional lethal mutations in the genes involved in the formation of the T4 head have been re-examined. If the product of genes 20 or 40 is defective, open-ended tubular structures (polyheads) are synthesized. Infection with mutants in gene 22 leads to the formation of polyheads, about 70% of which consist of two or more concentric layers (multilayered polyheads). Cells infected with mutants in gene 21 and 24 yield a mixed burst of capsid-like particles (τ-particles) and polyheads, many of which have hemispherical caps.
Two structures related to the head of bacteriophage T4, polyheads and τ-particles, are shown by electron microscopy to contain an internal component defined as a core. These cores can show a high degree of organization.Multilayered polyheads are described which contain a normal sized core.The dark polyhedral bodies seen in sections and previously referred to as DNA “condensates” were reinvestigated, and were found to have a visible membrane. However, this membrane is very fragile and does not survive lysis of the cell in a recognizable form.The appearance of thin sections of T4 heads obtained after different fixation methods suggests that the “less dense areas,” which can be observed when fixed under conditions found to be inadequate for DNA fixation, are likely to be artifacts and therefore cannot be used alone as proof for the presence of a core.We conclude that the present information from morphological, genetic, and biochemical experiments strongly suggests that the observed cores have a morphopoietic role. We discuss the structure and possible mechanism of action of such a core.
In reply to recent criticism Professor Commoner discusses current evidence in support of his conclusion that the Watson-Crick theory is an inadequate explanation of inheritance.
The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T4amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.
Fundamental Techniques of Virology
- J. V. Maizel
Fundamental Techniques of Virology (edit
- J V Maizel
- JV Maizel