Dynamics in the activity of peroxidase and its isoforms in leaves of different apple cultivars

Background. Various approaches are used for identification of the most resistant fruit crop cultivars, including the analysis of different physiological and biochemical indicators. In Krasnodar Territory, Russia, one of the major stressors in summer is the hydrothermal stress. Drought and heat lead to an oxidative stress, as reactive oxygen species are produced in plant cells. Plants respond to oxidative damage by activating antioxidant enzymes, such as superoxide dismutase, catalase, and various peroxidases. Peroxidase is able to decompose hydrogen peroxide. Peroxidase activity was calculated under natural summertime changes in the hydrothermal pattern (control) and in simulated high-temperature conditions.Materials and methods. Three apple cultivars of Russian breeding, ‘Fortuna’, ‘Soyuz’ and ‘Prikubanskoe’, and cv. ‘Ligol’ of Polish origin were studied. In the summers of 2018–2019, their leaf samples were analyzed to assess peroxidase activity and its isozyme composition under control and stress conditions. Native electrophoresis in polyacrylamide gel was used for separation of peroxidase isoforms. Malondialdehyde content was measured to identify oxidative stress levels in apple leaves.Results. The tested indicators demonstrated a high degree of heterogeneity induced by both cultivar specificity and seasonal weather dynamics. Peroxidase isoforms with a molecular weight of 70 to 60 kDa, characterized by the maximum level of variability (1–4 isoforms), were isolated. Two other groups included 1–3 isoforms with a molecular weight of ~130–100 kDa, and one with a molecular weight of ~55 kDa. The highest enzyme activity was found in cvs. ‘Fortuna’ and ‘Soyuz’ in July 2018, the hottest month during the period of research. Under simulated conditions, the triploid cultivar ‘Soyuz’ was least susceptible to the stress impact.

Proteasomes in Patient Rectal Cancer and Different Intestine Locations: Where Does Proteasome Pool Change?

A special problem in the surgery of rectal cancer is connected with a need for appropriate removal of intestine parts, along with the tumor, including the fragment close to the sphincter. To determine the length of fragments to remove, it is necessary to reveal areas without changes in molecule functioning, specific for tumor. The purpose of the present study was to investigate functioning the proteasomes, the main actors in protein hydrolysis, in patient rectal adenocarcinoma and different intestine locations. Chymotrypsin-like and caspase-like activities, open to complex influence of different factors, were analyzed in 43–54 samples by Suc-LLVY-AMC- and Z-LLE-AMC-hydrolysis correspondingly. Both activities may be arranged by the decrease in the location row: cancer→adjacent tissue→proximal (8–20 cm from tumor) and distal (2 and 4 cm from tumor) sides. These activities did not differ noticeably in proximal and distal locations. Similar patterns were detected for the activities and expression of immune subunits LMP2 and LMP7 and expression of 19S and PA28αβ activators. The largest changes in tumor were related to proteasome subtype containing LMP2 and PA28αβ that was demonstrated by native electrophoresis. Thus, the results indicate a significance of subtype LMP2-PA28αβ for tumor and absence of changes in proteasome pool in distal fragments of 2–4 cm from tumor.

Supramolecular associations between atypical oxidative phosphorylation complexes of Euglena gracilis

In vivo associations of respiratory complexes forming higher supramolecular structures are generally accepted nowadays. Supercomplexes (SC) built by complexes I, III and IV and the so-called respirasome (I/III2/IV) have been described in mitochondria from several model organisms (yeasts, mammals and green plants), but information is scarce in other lineages. Here we studied the supramolecular associations between the complexes I, III, IV and V from the secondary photosynthetic flagellate Euglena gracilis with an approach that involves the extraction with several mild detergents followed by native electrophoresis. Despite the presence of atypical subunit composition and additional structural domains described in Euglena complexes I, IV and V, canonical associations into III2/IV, III2/IV2 SCs and I/III2/IV respirasome were observed together with two oligomeric forms of the ATP synthase (V2 and V4). Among them, III2/IV SC could be observed by electron microscopy. The respirasome was further purified by two-step liquid chromatography and showed in-vitro oxygen consumption independent of the addition of external cytochrome c.

Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

Anti-glycation study of hydro-alcohol and aqueous extracts of Moroccan plant species

Inhibition of advanced glycation end products (AGEs) and free radicals generated during diabetes represents a major therapeutic target in the prevention and treatment of diabetic complications. Natural molecules present in fruits, vegetables and herbs and which are usually safe for human consumption, could represent a strong glycation inhibitor. Anti-glycation effect of nine plant species used in traditional medicine has been evaluated after extraction by hot (EAC) or cold (EAF) maceration and by ethanol (EE). Anti-glycation activity performed on a model system of bovine serum albumin, and methylglyoxal was measured by fluorescence and native electrophoresis. Total phenolic and flavonoid contents were assessed as well. Except for Sesamum indicum, all the species studied have an Anti-glycation effect. The highest effect was recorded in Laurus nobilis and was dose-dependent, inhibiting both formations of Amadori products and fluorescent AGEs. HPLC analysis revealed a richness of Laurus nobilis EE in phenolic compounds such as quercetin, vanillin and gallic acid. A strong correlation was registered between antioxidant power and phenolic/flavonoid content. In contrast, there was no correlation between antioxidant and anti-glycation power. Phenolic and flavonoid compounds were strongly involved in the observed anti-glycation effect. However, the anti-glycation activity obtained is probably attributed to non-antioxidant compounds.