Tat-malate dehydrogenase fusion protein protects neurons from oxidative and ischemic damage by reduction of reactive oxygen species and modulation of glutathione redox system.

Malate dehydrogenase (MDH) plays an important role in the conversion of malate to oxaloacetate during the tricarboxylic acid cycle. In this study, we examined the role of cytoplasmic MDH (MDH1) in hydrogen peroxide (H2O2)-induced oxidative stress in HT22 cells and ischemia-induced neuronal damage in the gerbil hippocampus. The Tat-MDH1 fusion protein was constructed to enable the delivery of MDH1 into the intracellular space and penetration of the blood-brain barrier. Tat-MDH1, but not MDH1 control protein, showed significant cellular delivery in HT22 cells in a concentration- and time-dependent manner and gradual intracellular degradation in HT22 cells. Treatment with 4 µM Tat-MDH1 significantly ameliorated 200 µM H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation in HT22 cells. Transient increases in MDH1 immunoreactivity were detected in the hippocampal CA1 region 6-12 h after ischemia, but MDH1 activity significantly decreased 2 days after ischemia. Supplementation of Tat-MDH1 immediately after ischemia alleviated ischemia-induced hyperlocomotion and neuronal damage 1 and 4 days after ischemia. In addition, treatment with Tat-MDH1 significantly ameliorated the increases in hydroperoxides, lipid peroxidation, and reactive oxygen species 2 days after ischemia. Tat-MDH1 treatment maintained the redox status of the glutathione system in the hippocampus 2 days after ischemia. These results suggest that Tat-MDH1 exerts neuroprotective effects by reducing oxidative stress and maintaining glutathione redox system in the hippocampus.

Dihydromyricetin alters myosin heavy chain expression via AMPK signaling pathway in porcine myotubes.

Dihydromyricetin (DHM) has attracted wide concern for its excellent biological function and pharmacological activities and was reported to have a positive effect on skeletal muscle insulin resistance, slow-twitch fibers expression and AMPK signaling. Thus, we took porcine myotubes derived from skeletal muscle satellite cells as the object to investigate the effects of DHM on myosin heavy chain (MyHC) expression and its mechanism in this study. Data showed that DHM up-regulated protein expression of MyHC I and down-regulated the protein expression of MyHC IIb, accompanied by an increase of MyHC I mRNA level and a decrease of MyHC IIb mRNA level. Besides, DHM increased the activities of malate dehydrogenase and succinic dehydrogenase and reduced lactate dehydrogenase activity. AMP-activated protein kinase (AMPK) was phosphorylated and AMPKα1 mRNA level was increased by DHM. The AMPK signaling-related factors including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), sirtuin1 (Sirt1), nuclear respiratory factor 1 (NRF1), and phospho-calmodulin-dependent protein kinase kinase-ß (p-CaMKKß) were increased by DHM. Inhibition of the AMPK signaling by compound C and AMPKα1 siRNA significantly attenuated the effects of DHM on expressions of MyHC I, MyHC IIb, PGC-1α and Sirt1. As a whole, DHM increased MyHC I expression and decreased MyHC IIb expression by the AMPK signaling.

Induction of systemic immunity through nasal-associated lymphoid tissue (NALT) of mice intranasally immunized with Brucella abortus malate dehydrogenase-loaded chitosan nanoparticles.

Infection with Brucella abortus causes contagious zoonosis, brucellosis, and leads to abortion in animals and chronic illness in humans. Chitosan nanoparticles (CNs), biocompatible and nontoxic polymers, acts as a mucosal adjuvant. In our previous study, B. abortus malate dehydrogenase (Mdh) was loaded in CNs, and it induced high production of pro-inflammatory cytokines in THP-1 cells and systemic IgA in BALB/C mice. In this study, the time-series gene expression analysis of nasal-associated lymphoid tissue (NALT) was performed to identify the mechanism by which Mdh affect the target site of nasal immunization. We showed that intranasal immunization of CNs-Mdh reduced cell viability of epithelial cells and muscle cells at first 1 h, then induced cellular movement of immune cells such as granulocytes, neutrophils and lymphocytes at 6h, and activated IL-6 signaling pathway at 12h within NALT. These activation of immune cells also promoted signaling pathway for high-mobility group box 1 protein (HMGB1), followed by the maturation of DCs required for mucosal immunity. The CNs also triggered the response to other organism and inflammatory response, showing it is immune-enhancing adjuvant. The ELISA showed that significant production of specific IgA was detected in the fecal excretions and genital secretions from the CNs-Mdh-immunized group after 2 weeks-post immunization. Collectively, these results suggest that B. abortus Mdh-loaded CNs triggers activation of HMGB1, IL-6 and DCs maturation signaling within NALT and induce production of systemic IgG and IgA.

Effects of in ovo administration of DHEA on lipid metabolism and hepatic lipogenetic genes expression in broiler chickens during embryonic development.

In order to study the mechanism of DHEA (Dehydroepiandrosterone) in reducing fat in broiler chickens during embryonic development, fertilized eggs were administrated with DHEA before incubation and its effect on lipid metabolism and expression of hepatic lipogenetic genes was investigated. The mRNA levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100 (apoB100) and sterol regulator element binding protein-1c (SREBP-1c) were determined using real time quantitative PCR. Samples of livers were collected from the chickens on days 9, 14, and 19 of embryonic development as well as at hatching. Blood samples were extracted on days 14, 19 of incubation and at hatching. The results showed that DHEA decreased the concentration of triacyglycerol in the blood and the content in liver, and the mRNA levels of ACC, FAS, ME, SREBP-1c and apoB. This suggested that DHEA decreased the expression of hepatic lipogenetic genes and suppressed triglycerols transport, by which it reduced the deposition of fat in adipose tissue in broiler chickens during embryonic development and hatching.

Endosulfan effects on muscle malate dehydrogenase of the freshwater catfish Clarias batrachus.

The effects of a sublethal concentration of an organochlorine pesticide endosulfan were investigated on crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) in the muscle of the freshwater catfish Clarias batrachus. Endosulfan treatment reduced significantly the activity and the specific activity of cMDH and mMDH but had no effect on total cytoplasmic and mitochondrial protein contents. This indicates a decline in the efficiency of aerobic energy metabolism in endosulfan-treated fish. The polyacrylamide gel electrophoresis showed two distinct isoforms (C1 and C2) of cMDH and only one form of mMDH. The inhibition produced by endosulfan in vivo was of mixed noncompetitive/uncompetitive type for crude as well as purified cMDH and mMDH. However, in vitro inhibition was of simple noncompetitive and mixed competitive/noncompetitive type for purified cMDH and mMDH, respectively. Citrate was found to be an uncompetitive inhibitor of cMDH and mixed noncompetitive/uncompetitve inhibitor of mMDH. The inhibitory patterns were modulated by endosulfan. These results demonstrate inhibitory effects of endosulfan on skeletal muscle MDH of the freshwater catfish Clarias batrachus and inhibition effects are mediated through enzyme/substrate/endosulfan complexing.

Bound substrate polypeptides can generally stabilize the tetradecameric structure of Cpn60 and induce its reassembly from monomers.

We demonstrate that the previously observed stabilizing effect by the enzyme rhodanese of the oligomeric structure of Cpn60 is general and can be provided by six other proteins that can interact with Cpn60. All these substrate proteins, which include examples that are monomeric, as well as oligomeric polypeptides in their native states, were shown previously to be assisted in their refolding by the chaperonin. Strikingly, during the disassembly of Cpn60 in the presence of any of the substrate proteins, significant amounts of intermediates were detected. Furthermore, unfolded substrate proteins induce the reassembly of tetradecameric Cpn60 from monomers, and binding of each substrate protein stabilizes Cpn60 quaternary structure.

Apparent inhibition of ATP citrate lyase by L-glutamate in vitro is due to the presence of glutamine synthetase.

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.