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.

Serum protein complex profiling reveals heterogeneity of Balanced constitutional population in traditional Chinese medicine through blue native polyacrylamide gel electrophoresis.

BACKGROUND: Pulse-taking is widely used for diagnosis and treatment in traditional Chinese medicine (TCM), and protein complexes in serum perform various biological functions. The Balanced constitution is one of the major constitutions in TCM, people with Balanced constitution can also share some common characteristics with unbalanced constitution types. METHODS: Blue native polyacrylamide gel electrophoresis (BN-PAGE) was applied to the serum of 25 people with balanced constitutions. The patterns of the protein complexes could be recognized according to the number, molecular weight, and intensity of the gel bands. All of the individual bands from these patterns were cut and in-gel-digested with trypsin, followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis for protein identification and biological function analysis. RESULTS: The protein complex patterns were roughly categorized as type A and B with high stability and reproducibility, and there were 15 and 16 gel bands in type A and type B, respectively. Among the 25 serum samples, 14 belonged to type A, and 11 belonged to type B. High-abundance proteins significantly decreased from 99% to 44% after BN-PAGE separation. The unique proteins in type A were mainly related to lipid metabolism, while the unique proteins in type B were involved in biological processes related to immune response and inflammatory regulation. The Qi-deficiency constitution converted score of type A was higher than that of type B, while the Damp-heat constitution converted score of type A was lower than that of type B. CONCLUSIONS: Our study provided an objective reference for diagnosis and prognosis, which might lay a foundation for establishing the characteristic protein complex spectra of all of the TCM constitutions.

Measurement of S-Nitrosoglutathione Reductase Activity in Plants.

S-nitrosation as a redox-based posttranslational modification of protein cysteine has emerged as an integral part of signaling pathways of nitric oxide across all types of organisms. Protein S-nitrosation status is controlled by two key mechanisms: by direct denitrosation performed by the thioredoxin/thioredoxin reductase system, and in an indirect way mediated by S-nitrosoglutathione reductase (GSNOR). GSNOR, which has been identified as a key component of S-nitrosothiols catabolism, catalyzes an irreversible decomposition of abundant intracellular S-nitrosothiol, S-nitrosoglutathione (GSNO) to oxidized glutathione using reduced NADH cofactor. In plants, GSNOR has been shown to play important roles in plant growth and development and plant responses to abiotic and biotic stress stimuli. In this chapter, optimized protocols of spectrophotometric measurement of GSNOR enzymatic activity and activity staining in native polyacrylamide gels in plant GSNOR are presented.

Direct Transfection of Fatty Acid Conjugated siRNAs and Knockdown of the Glucose-Regulated Chaperones in Prostate Cancer Cells.

The emerging field of RNAi nanotechnology has led to rapid advances in the applications of siRNAs in chemical biology, medicinal chemistry, and biotechnology. In our RNAi approach, bioconjugation of linear, V-, and Y-shaped RNA templates were designed using a series of saturated and unsaturated fatty acids to improve cell uptake and knockdown efficacy of the oncogenic glucose regulated proteins (GRPs) in prostate (PC-3) cancer cells. An optimized HCTU-coupling procedure was developed for tagging variable saturated and unsaturated fatty acids onto the 5'-ends of linear and V-shaped RNA templates that were constructed by semiautomated solid phase RNA synthesis. Hybridization and self-assembly of complementary strands yielded linear, V-, and Y-shaped fatty acid-conjugated siRNAs which were characterized by native PAGE. CD spectroscopy confirmed their A-type helix conformations. RP IP HPLC provided trends in amphiphilic properties, whereas DLS and TEM confirmed multicomponent self-assembled structures that were prone to aggregation. Subsequently, the fatty acid conjugated siRNA bioconjugates were tested for their RNAi activity by direct transfection within PC-3 cells known to overexpress oncogenic GRP activity. The siRNA bioconjugates with sense strand modifiers provided more potent GRP knockdown relative to the antisense modified siRNAs, but to a lesser extent when compared to the unconjugated siRNA controls that were transfected with the commercial Trans-IT X2 dynamic delivery system. Flow cytometry revealed that the latter may be at least in part attributed to limited cell uptake of the fatty acid conjugated siRNAs. Nonetheless, these new constructs represent an entry point in modifying higher-order siRNA constructs that may lead to the generation of more efficient siRNA bioconjugates for screening important oncogene targets and for cancer gene therapy applications.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

Physiological and proteomic analysis in chloroplasts of Solanum lycopersicum L. under silicon efficiency and salinity stress.

Tomato plants often grow in saline environments in Mediterranean countries where salt accumulation in the soil is a major abiotic stress that limits its productivity. However, silicon (Si) supplementation has been reported to improve tolerance against several forms of abiotic stress. The primary aim of our study was to investigate, using comparative physiological and proteomic approaches, salinity stress in chloroplasts of tomato under silicon supplementation. Tomato seedlings (Solanum lycopersicum L.) were grown in nutrient media in the presence or absence of NaCl and supplemented with silicon for 5 days. Salinity stress caused oxidative damage, followed by a decrease in silicon concentrations in the leaves of the tomato plants. However, supplementation with silicon had an overall protective effect against this stress. The major physiological parameters measured in our studies including total chlorophyll and carotenoid content were largely decreased under salinity stress, but were recovered in the presence of silicon. Insufficient levels of net-photosynthesis, transpiration and stomatal conductance were also largely improved by silicon supplementation. Proteomics analysis of chloroplasts analyzed by 2D-BN-PAGE (second-dimensional blue native polyacrylamide-gel electrophoresis) revealed a high sensitivity of multiprotein complex proteins (MCPs) such as photosystems I (PSI) and II (PSII) to the presence of saline. A significant reduction in cytochrome b6/f and the ATP-synthase complex was also alleviated by silicon during salinity stress, while the complex forms of light harvesting complex trimers and monomers (LHCs) were rapidly up-regulated. Our results suggest that silicon plays an important role in moderating damage to chloroplasts and their metabolism in saline environments. We therefore hypothesize that tomato plants have a greater capacity for tolerating saline stress through the improvement of photosynthetic metabolism and chloroplast proteome expression after silicon supplementation.

[In vitro study on gastrointestinal absorption of FITC labeled pilose antler protein extraction].

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.

In vitro study on gastrointestinal absorption of FITC labeled pilose antler protein extraction / 药学学报

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.

Listeria seeligeri Gene Encoding Heme-containing Catalase Produces No Activity in Streptococcus pneumoniae

Streptococcus pneumoniae is a facultative anaerobe lacking catalase enzyme and requires exogenous catalase supplemented to culture media for aerobic growth. We introduced a catalase gene (kat) of Listeria seeligeri into S. pneumoniae and tried to see if this listerial kat gene was expressed within the pneumococcal host. To clone the listerial kat gene in the pneumococcal chromosome, a non-replicating plasmid pAHA-LSt3, along with its original promoter region was used for integration the chromosome via homologous recombination. One of three resulting transformants was confirmed to contain the kat gene and designated as EHS2. In addition, the kat gene was subcloned in Escherichia coli in frame to the lac promoter of a shuttle vector to generate pDL-Kat, which was subsequently used for pneumococcal transformation. Four identical recombinants were identified to contain the plasmid with the kat gene. By performing RT-PCR, it was observed that the listerial kat gene was indeed transcribed within pneumococcal recombinants from its original promoter in the chromosome of EHS2 and from the lac promoter in the plasmid pDL-Kat. In contrast to the E. coli kat+ recombinants, however, the pneumococcal kat+ recombinants failed to reveal any catalase activities detectable by ferricyanide staining on non-denaturing PAGE. When the pDL-Kat plasmid DNA purified from pneumococci was allowed to transform E. coli again, many kat+ recombinants were obtained, ruling out the possibility of the defective kat E. coli transformants gene within pneumococci. The observation that the listerial kat gene in pneumococci was unable to produce the functional catalase enzyme, which requires a heme group at its active site and a cofactor NADPH, suggests pneumococcal defect in heme production.