Nondenaturing Polyacrylamide Gel Electrophoresis Research Articles

Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

Comparative Analysis of Catalase Activity in Plants: Spectrophotometry and Native PAGE Approaches.

Catalase, a pivotal enzyme in plant antioxidative defense mechanisms, plays a crucial role in detoxifying hydrogen peroxide, a reactive oxygen species (ROS). In this chapter, a comparative analysis of catalase activity was conducted using two distinct methodologies: spectrophotometry and non-denaturing polyacrylamide gel electrophoresis (PAGE). The spectrophotometric approach allowed the quantification of catalase activity by measuring the breakdown rate of hydrogen peroxide, while native PAGE enabled the separation and visualization of catalase isozymes, based on their native molecular weight and charge characteristics, and specific staining assay. Both methods provide valuable insights into catalase activity, offering complementary information on the enzyme's functional diversity and distribution within different plant tissues. This study integrates different techniques, previously described, to comprehensively elucidate the role of catalase in plant metabolism. Furthermore, it provides the possibility of obtaining a holistic understanding of antioxidant defense mechanisms by considering both total activity and isoenzyme distribution of catalase enzyme.

Identification of Superoxide Dismutase (SOD) Isozymes in Plant Tissues.

Superoxide and hydrogen peroxide are reactive oxygen species (ROS) involved in the oxidation of multiple biological molecules and the signaling processes during plant growth and stress response. Thus, control of ROS is fundamental for cell survival and development, with superoxide dismutase (EC 1.15.1.1, SOD) being one of the main enzymes involved. Different isoforms of SOD catalyze the dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2) and oxygen (O2), such as Mn-SODs, Cu,Zn-SODs, and Fe-SODs. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) combined with a specific staining method for SOD activity, the protocol describes the identification of different SOD isozymes, based on their differential inhibition by KCN and H2O2, in different organs and plant species such as pea (Pisum sativum L.) leaves and pepper (Capsicum annuum L.) fruits.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity.

Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression. Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura×Pisifera (D×P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279. The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

NADP-Dependent Malic Enzyme Genes in Sweet Pepper Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO).

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50-75% ammonium-sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.

Isoenzymatic Pattern of Hydrogen Sulfide (H2S)-Generating L-Cysteine Desulfhydrase (LCD) in Arabidopsis thaliana Seedlings: Effect of Nitric Oxide (NO) and H2S

In higher plants, hydrogen sulfide (H2S) is a recognized signaling molecule that performs multiple regulatory functions. The enzyme L-cysteine desulfhydrase (LCD) catalyzes the conversion of L-cysteine (L-Cys) to pyruvate and ammonium with the concomitant generation of H₂S, and it is considered one of the main sources of H2S in plants. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a specific assay for LCD activity, this study aims to identify the potential LCD isozymes in wild-type Arabidopsis thaliana seedlings of 16 days old grown under in vitro conditions, and to evaluate the potential impact of nitric oxide (NO) and H2S on these LCD isozymes. For this purpose, an Atnoa1 mutant characterized to have a low endogenous NO content as well as the exogenous application of H2S were used. Five LCD isozymes were detected, with LCD IV being the isozyme that has the highest activity. However, the LCD V activity was the only one that was positively modulated in the Atnoa1 mutants and by exogenous H2S. To our knowledge, this is the first report showing the different LCD isozymes present in Arabidopsis seedlings and how their activity is affected by NO and H2S content.

H2S-Generating Cytosolic L-Cysteine Desulfhydrase and Mitochondrial D-Cysteine Desulfhydrase from Sweet Pepper (Capsicum annuum L.) Are Regulated During Fruit Ripening and by Nitric Oxide.

Aims: Pepper fruit is a horticultural product worldwide consumed that has great nutritional and economic relevance. Besides the phenotypical changes that undergo pepper fruit during ripening, there are many associated modifications at transcriptomic, proteomic, biochemical, and metabolic levels. Nitric oxide (NO) and hydrogen sulfide (H2S) are recognized signal molecules that can exert regulatory functions in diverse plant processes. This study aims at analyzing the interrelationship between NO and H2S during fruit ripening. Results: Our data indicate that the H2S-generating cytosolic L-cysteine desulfhydrase (LCD) and the mitochondrial D-cysteine desulfhydrase (DCD) activities are downregulated during ripening but this effect was reverted after NO treatment of fruits. Innovation and Conclusion: Using as a model the non-climacteric pepper fruits at different ripening stages and under an NO-enriched atmosphere, the activity of the H2S-generating LCD and DCD was analyzed. LCD and DCD activities were downregulated during ripening, but this effect was reverted after NO treatment of fruits. The analysis of LCD activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) allowed identifying three isozymes designated CaLCD I to CaLCD III, which were differentially modulated by NO and strictly dependent on pyridoxal 5'-phosphate (PLP). In vitro analyses of green fruit samples in the presence of different compounds including NO donors, peroxynitrite (ONOO-), and reducing agents such as reduced glutathione (GSH) and L-cysteine (L-Cys) triggered an almost 100% inhibition of CaLCD II and CaLCD III. This redox adaptation process of both enzymes could be cataloged as a hormesis phenomenon. The protein tyrosine (Tyr) nitration (an NO-promoted post-translational modification) of the recombinant LCD was corroborated by immunoblot and by mass spectrometry (MS) analyses. Among the 11 Tyr residues present in this enzyme, MS of the recombinant LCD enabled us to identify that Tyr82 and Tyr254 were nitrated by ONOO-, this occurring near the active center on the enzyme, where His237 and Lys260 together with the cofactor PLP are involved. These data support the relationship between NO and H2S during pepper fruit ripening, since LCD and DCD are regulated by NO during this physiological event, and this could also be extrapolated to other plant species.

High Molecular Weight α-Galactosidase from the Novel Strain Aspergillus sp. D-23 and Its Hydrolysis Performance

Aspergillus sp. D-23 was obtained by ultraviolet-diethyl sulfate (UV-DES) compound mutagenesis from Aspergillus sp. C18 that the α-galactosidase was purified from. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), the purified enzyme demonstrated apparent homogeneity. The monomeric α-galactosidase’s native molecular weight was 125 kDa. The optimal temperature of α-galactosidase was 65 °C, and 75% of the initial enzyme activity could be maintained between 45 and 55 °C. Its optimal pH was 5.0 with good pH stability. After incubating for 2 h at pH 3.0–8.0, it could retain more than 80% of its original activity. Different concentrations of metal ions had different effects on the α-galactosidase activity. High concentrations of Cu2+ could strongly inhibit enzyme activity and low concentrations of Fe2+ could promote enzyme activity. Additionally, as shown by thin layer chromatography and high-performance liquid chromatography, the enzyme also had good hydrolysis ability, which could efficiently hydrolyze melibiose and raffinose by more than 95%. Therefore, these excellent characteristics could make α-galactosidase a good candidate for the food and feed industries.

Analysis of Plant L-Cysteine Desulfhydrase (LCD) Isozymes by Non-denaturing Polyacrylamide Gel Electrophoresis.

Hydrogen sulfide (H2S) is a signaling molecule that achieves different regulatory functions in animal and plant cells. The cytosolic enzyme L-cysteine desulfhydrase (LCD; EC 4.4.1.28) catalyzes the conversion of cysteine (L-Cys) to pyruvate and ammonium with the concomitant generation of H2S, this enzyme being considered one of the main sources of H2S in higher plants. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a specific assay for LCD activity, the present protocol allows identifying diverse LCD isozymes present in different organs (roots, shoots, leaves, and fruits) and plant species including pea, garlic, Arabidopsis, and pepper.

Iron Accumulation in Ferritin.

Understanding the iron accumulation process in ferritin protein nanocages has remained a centerpiece in the field of iron biochemistry/biomineralization, which ultimately has implications in health and diseases. Although mechanistic differences of iron acquisition and mineralization exist in the superfamily of ferritins, we describe the techniques that can be used to investigate the accumulation of iron in all the ferritin proteins by in vitro iron mineralization process. In this chapter, we report that the non-denaturing polyacrylamide gel electrophoresis coupled with Prussian blue staining (in-gel assay) can be useful to investigate the iron-loading efficiency in ferritin protein nanocage, by estimating the relative amount of iron incorporated inside it. Similarly, the absolute size of the iron mineral core and the amount of total iron accumulated inside its nanocavity can be determined by using transmission electron microscopy and spectrophotometry, respectively.

Lipoxygenase (LOX) in Sweet and Hot Pepper (Capsicum annuum L.) Fruits during Ripening and under an Enriched Nitric Oxide (NO) Gas Atmosphere

Lipoxygenases (LOXs) catalyze the insertion of molecular oxygen into polyunsaturated fatty acids (PUFA) such as linoleic and linolenic acids, being the first step in the biosynthesis of a large group of biologically active fatty acid (FA)-derived metabolites collectively named oxylipins. LOXs are involved in multiple functions such as the biosynthesis of jasmonic acid (JA) and volatile molecules related to the aroma and flavor production of plant tissues, among others. Using sweet pepper (Capsicum annuum L.) plants as a model, LOX activity was assayed by non-denaturing polyacrylamide gel electrophoresis (PAGE) and specific in-gel activity staining. Thus, we identified a total of seven LOX isozymes (I to VII) distributed among the main plant organs (roots, stems, leaves, and fruits). Furthermore, we studied the FA profile and the LOX isozyme pattern in pepper fruits including a sweet variety (Melchor) and three autochthonous Spanish varieties that have different pungency levels (Piquillo, Padrón, and Alegría riojana). It was observed that the number of LOX isozymes increased as the capsaicin content increased in the fruits. On the other hand, a total of eight CaLOX genes were identified in sweet pepper fruits, and their expression was differentially regulated during ripening and by the treatment with nitric oxide (NO) gas. Finally, a deeper analysis of the LOX IV isoenzyme activity in the presence of nitrosocysteine (CysNO, a NO donor) suggests a regulatory mechanism via S-nitrosation. In summary, our data indicate that the different LOX isozymes are differentially regulated by the capsaicin content, fruit ripening, and NO.

Development of ILP-markers for Aegilops tauschii and their application in molecular genetic analysis

To develop marker systems based on the study of intron length polymorphism of various genes (ILP) in Ae. tauschii, to test the possibility of their use for genetic differentiation of Ae. tauschii and Ae. biuncialis. Methods. The NCBI database was used to obtain EST sequences, online tools CD-HIT, BLAST and Primer3Plus (for the development of primers). The polymerase chain reaction (PCR) method with the developed primers was done. Amplified fragments were separated by non-denaturing polyacrylamide gel electrophoresis and stained with silver. Results. Molecular markers Aet_ILP1, Aet_ILP6 were developed and tested for different genotypes of Ae. tauschii and Ae. biuncialis. No intraspecific differentiation was observed in all studied samples, but amplicons of introns of different species differed significantly. It was also found that the studied species had one copy of the HO222074.1 gene and 1 to 3 copies of the CX244643.1 gene. Conclusions. The obtained results indicate a low level of intraspecific variability of the developed ILP markers and the possibility of their use for interspecific differentiation of Ae. tauschii and Ae. biuncialis.

Analysis of the Protein Content of Nutritional Shakes

High protein shakes have become increasingly popular as nutritional supplements for adolescents, adults, and senior citizens. They are heavily advertised on television and in print media and are available in grocery and health food stores, at pharmacies, and from online distributors. In this project, students use colorimetric assays to determine the protein concentration of a particular product they bring to class and compare the results to the nutritional label. Students then use denaturing and nondenaturing polyacrylamide gel electrophoresis to characterize the protein composition of their protein shakes. These experiments promote critical thinking about common scientific procedures and should be of interest to both secondary school and college students.

Hemopexin as a Potential Binding Partner of Arginine-Rich Cell-Penetrating Peptides in Serum.

Serum proteins affect the in vivo fate and cellular uptake of arginine-rich cell-penetrating peptides (CPPs) and drugs delivered by CPPs. Although the binding of CPPs to serum proteins may possibly reduce their cellular uptake to some extent, it may also prolong their circulation half-life in vivo. We aimed to identify novel binding proteins of arginine-rich CPPs in serum to better understand their in vivo fate and develop more sophisticated drug delivery systems using CPPs. Isothermal titration calorimetry analysis suggests that albumin, the most abundant protein in serum, binds to d-forms of oligoarginine; however, the dissociation constants are several tens of a micromolar. Candidate proteins with the potential of binding to arginine-rich CPPs in serum were then explored using nondenaturing polyacrylamide gel electrophoresis followed by mass spectrometry analysis. Studies using fluorescence correlation spectroscopy determined hemopexin as a potential binding partner of d-forms of arginine-rich CPPs, including d-octaarginine (r 8) and the d-form of the peptide, corresponding to HIV-1 Rev (34-50), with dissociation constants of submicromolar to micromolar range. Using flow cytometry and a split-luciferase-based system, the promotion effect of hemopexin on the total cellular uptake and cytosolic localization of cargos conjugated with these CPPs was confirmed. Therefore, this study elucidated hemopexin as a potential binding partner of d-arginine-rich CPPs that may affect their in vivo fate and cellular uptake.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Antiproliferative Effects of the Aptamer d(GGGT)4 and Its Analogues with an Abasic-Site Mimic Loop on Different Cancer Cells.

In this paper, we study the T30923 antiproliferative potential and the contribution of its loop residues in six different human cancer cell lines by preparing five T30923 variants using the single residue replacement approach of loop thymidine with an abasic site mimic (S). G-rich oligonucleotides (GRO) show interesting anticancer properties because of their capability to adopt G-quadruplex structures (G4s), such as the G4 HIV-1 integrase inhibitor T30923. Considering the multi-targeted effects of G4-aptamers and the limited number of cancer cell lines tested, particularly for T30923, it should be important to find a suitable tumor line, in addition to considering that the effects also strictly depend on G4s. CD, NMR and non-denaturating polyacrylamide gel electrophoresis data clearly show that all modified ODNs closely resemble the dimeric structure of parallel G4s’ parent aptamer, keeping the resistance in biological environments substantially unchanged, as shown by nuclease stability assay. The antiproliferative effects of T30923 and its variants are tried in vitro by MTT assays, showing interesting cytotoxic activity, depending on time and dose, for all G4s, especially in MDA-MB-231 cells with a reduction in cell viability approximately up to 30%. Among all derivatives, QS12 results are the most promising, showing more pronounced cytotoxic effects both in MDA-MB-231 and Hela cells, with a decrease in cell viability from 70% to 60%. In summary, the single loop residue S substitution approach may be useful for designing antiproliferative G4s, considering that most of them, characterized by single residue loops, may be able to bind different targets in several cancer cell pathways. Generally, this approach could be of benefit by revealing some minimal functional structures, stimulating further studies aimed at the development of novel anticancer drugs.

Exploring a peptide nucleic acid-based antisense approach for CD5 targeting in chronic lymphocytic leukemia.

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

Biochemically prepared C-reactive protein conformational states differentially affect C1q binding

C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections. Due to a correlation of CRP to a vast number of disease states, CRP is a well-studied protein in medical literature with over 16000 references in PubMed [1]. However, the biochemical and structural variations of CRP are not well understood in regards to their binding of complement immune response proteins. Conformations of CRP are thought to affect disease states differently, with a modified form showing neoepitopes and activating the complement immune response through C1q binding. In this work, we compare the unfolding of CRP using chemical denaturants and identify which states of CRP bind a downstream complement immune response binding partner (C1q). We used guanidine HCl (GndHCl), urea/EDTA, and 0.01% SDS with heat to perturb the pentameric state. All treatments give rise to a monomeric state in non-denaturing polyacrylamide gel electrophoresis experiments, but only treatment with certain concentrations of denaturant or dilute SDS with heat maintains CRP function with a key downstream binding partner, C1q, as measured by enzyme-linked immunosorbent assays. The results suggest that the final form of modified CRP and its ability to mimic biological binding is dependent on the preparation method.

Genetic polymorphism of white mistletoe (Viscum album L.) in Ukraine

Aim. The aim of the study was to establish genetic differences between V. album growing in different parts of Ukraine. Methods. White mistletoe samples collected in different regions of Ukraine were used in the study. The method of estimating the intron length polymorphism of β-tubulin genes was used. Amplified DNA fragments were fractionated by non-denaturing polyacrylamide gel electrophoresis and visualized by silver nitrate staining. Results. The genotypes of 91 white mistletoe plants were analyzed. DNA profiles of white mistletoe with a specific amplicons of β-tubulin gene introns were obtained, which allowed to differentiate the samples from each other. Fingerprinting data were used for cluster analysis and dendrogram construction. Conclusions. It was found that the analyzed mistletoe samples did not differ by geographical factor and were characterized by a low level of genetic diversity in the studied samples.
 Keywords: Viscum album L., intron length polymorphism, β-tubulin, genetic variability, Ukraine.