Effects of resveratrol on DLD and NDUFB9 decrease in frozen semen of Mongolian sheep.

Mongolian sheep are a breed of sheep in China known for their excellent cold and drought resistance. Sperm from Mongolian sheep are often cryopreserved to improve breeding outcomes. However, cryopreservation of sperm often results in issues such as reduced vitality and altered morphology. Therefore, the objective of this study was to investigate the impact of the cryoprotectant resveratrol on frozen sperm from Mongolian sheep, specifically examining its effects on key proteins during cryopreservation. In this study, sperm samples were obtained from three adult Mongolian rams and processed through semen centrifugation. The sperm motility parameters of Fresh Sperm Group (FR), Resveratrol added before freezing group (FF-Res), Resveratrol-free frozen sperm group (FT), and Resveratrol added after freeze-thawing group (FA-Res) were determined. The tandem mass tags (TMT) peptide labeling combined with LC-MS/MS was used for proteomic analysis of the total proteins in FR and FT groups. A total of 2651 proteins were identified, among which 41 proteins were upregulated and 48 proteins were downregulated after freezing. In-depth bioinformatics analysis of differentially abundant proteins (DAPs) revealed their close association with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation pathway. The energy-related protein dihydrolipoamide dehydrogenase (DLD) and the reactive oxygen species (ROS)-related protein NADH dehydrogenase 1 beta subcomplex subunit 9 (NDUFB9) exhibited significant decreases, indicating their potential role as key proteins contributing to reduced sperm vitality. The study demonstrated that the addition of resveratrol (RES) to semen could elevate the expression levels of DLD and NDUFB9 proteins. This study represents the pioneering proteomic analysis of Mongolian ram sperm before and after cryopreservation, establishing the significance of DLD and NDUFB9 as key proteins influencing the decline in vitality following cryopreservation of Mongolian ram sperm. These findings clarify that resveratrol can enhance the levels of DLD and NDUFB9 proteins in cryopreserved Mongolian ram sperm, consequently enhancing their vitality.

pH-Controlled Release of Antigens Using Mesoporous Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine.

The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 µg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.

Diaphorase catalyzed biotransformation of RDX via N-denitration mechanism.

Previously, we hypothesized that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be biotransformed by anaerobic sludge via three different routes: (1) direct ring cleavage via alpha-hydroxylation of a-CH(2) group, (2) reduction of one of the -NO(2) groups to -NO, (3) N-denitration prior to ring cleavage. The present study describes biotransformation of RDX via route 3 by a diaphorase (EC 1.8.1.4) from Clostridium kluyveri using NADH as electron donor. The removal of RDX was accompanied by the formation and accumulation of nitrite ion (NO(2)(-)), formaldehyde (HCHO), ammonium (NH(4)(+)), and nitrous oxide (N(2)O). None of the RDX-nitroso products were detected. The ring cleavage product methylenedinitramine was detected as the transient intermediate. Product stoichiometry showed that each reacted RDX molecule produced one nitrite ion and the product distribution gave a carbon (C) and nitrogen (N) mass balance of 91 and 92%, respectively, supporting the occurrence of a mono-denitration step prior to the ring cleavage and decomposition. Severe oxygen mediated inhibition (92% inhibition) of RDX biotransformation and superoxide dismutase-sensitive cytochrome c reduction indicated the potential involvement of an anion radical RDX(.-) prior to denitration. A comparative study between native- and apo-enzymes showed the possible involvement of flavin mononucleotide (FMN) in catalyzing the transfer of a redox equivalent (e/H(+)) from NADH to RDX to produce RDX(.-) responsible for secondary decomposition.

Superoxide-mediated release of iron from ferritin by some flavoenzymes.

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and release iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methyl-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.

Further studies of sulfoxide-reducing enzyme system.

Various kinds of flavoenzymes such as NADPH-cytochrome c reductase, NADH-cytochrome b5 reductase, xanthine oxidase, lipoamide dehydrogenase and NADH dehydrogenase supplemented with their electron donors exhibited the sulfoxide reductase activity in the presence of a partially purified soluble factor from guinea pig liver. The present study suggests that new electron transfer systems in which the soluble factor functions as an electron carrier coupled with flavoenzymes described above are responsible for the sulfoxide reduction.

Lipid peroxidation of erythrocyte membrane induced by lipoamide dehydrogenase in the presence of ADP-Fe3+.

Lipid peroxidation of rat erythrocyte membranes was induced by lipoamide dehydrogenase (LADH) (EC 1.8.1.4) in the presence of ADP-Fe3+. Superoxide dismutase (SOD) (EC 1.15.1.1) strongly inhibited the peroxidation reaction but catalase did not. Hydroxyl radical scavengers, mannitol and dimethylsulfoxide did not inhibit the lipid peroxidation. These results indicated that the lipid peroxidation was a superoxide (O2-)-dependent reaction, but the hydroxyl radical was not involved. ADP-Fe3+, in the presence of LADH, was reduced more rapidly under aerobic than anaerobic conditions and SOD under aerobic conditions strongly inhibited the iron reduction, indicating that O2- plays a predominant role in iron reduction. Hydrogen peroxide enhanced O2- generation by LADH, but the peroxidation reaction was not affected. In the presence of lipoamide, lipid peroxidation was also induced but the reactions were not inhibited by SOD. Evidently, the lipid peroxidation induced in the presence of lipoamide was O2(-)-independent. Dihydrolipoamide may be involved in the peroxidation reaction.