Proteomic investigation of changes in rat skeletal muscle after exercise-induced fatigue.

The mechanisms of exercise-induced fatigue have not been investigated using proteomic techniques, an approach that could improve our understanding and generate novel information regarding the effects of exercise. In this study, the proteom alterations of rat skeletal muscle were investigated during exercise-induced fatigue. The proteins were extracted from the skeletal muscle of SD rat thigh, and then analyzed by two-dimensional electrophoresis and PDQuest software. Compared to control samples, 10 significantly altered proteins were found in exercise samples, two of them were upregulated and eight of them were downregulated. These proteins were identified by MALDI TOF-MS. The two upregulated proteins were identified as MLC1 and myosin L2 (DTNB) regulatory light-chain precursors. The eight decreased proteins are Glyceraldehyde-3-phosphate Dehydrogenas (GAPDH); Beta enolase; Creatine kinase M chain (M-CK); ATP-AMP Transphosphorylase (AK1); myosin heavy chain (MHC); actin; Troponin I, fast-skeletal muscle (Troponin I fast-twitch isoform), fsTnI; Troponin T, fast-skeletal muscle isoforms (TnTF). In these proteins, four of the eight decreased proteins are related directly or indirectly to exercise induced fatigue. The other proteins represent diverse sets of proteins including enzymyes related to energy metabolism, skeletal muscle fabric protein and protein with unknown functions. They did not exhibit evident relationship with exercise-induced fatigue. Whereas the two identified increased proteins exhibit evident relationship with fatigue. These findings will help in understanding the mechanisms involved in exercise-induced fatigue.

Proteomic analysis of cerebral synaptosomes isolated from rat model of alzheimer's disease.

Alzheimer's disease (AD) is a common and devastating disease and there is no readily available biomarker to aid diagnosis or monitor progression of it. To further understand the pathogenic mechanism of AD, proteomic approach was used to study the cerebral synaptosomes proteins of rats injected with Abeta1-40. Compared with the untreated samples, 14 proteins were found apparently altered through 2-dimensional gel electrophoresis. 12 of them were down-regulated and 2 were up-regulated. Three proteins including alpha-2-globin chain, peptidyl-prolycis-trans isomerase A (PPIaseA) and cofilin-1 protein were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database query. Alpha-2-globin chain has not been shown to be associated with AD. PPIaseA and cofilin-1 protein are correlated with cell apoptosis and signaling. The altered proteins identified may help to understand the pathogenesis of AD.

Generation of SHMT2 knockout human embryonic stem cell line (WAe009-A-67) using CRISPR/Cas9 technique.

Serine hydroxymethyltransferase 2 (SHMT2), a catalytic enzyme playing an important role in aerobic cellular respiration and mitochondrial metabolism, might be pivotal in self-renewal and differentiation of human pluripotent stem cells. Herein, we used the CRISPR/Cas9 editing system to construct a homozygous SHMT2 knockout (SHMT2-KO) human embryonic stem cell (hESC) line, exhibiting a normal karyotype, colony morphology, and high expression levels of pluripotent proteins. Furthermore, SHMT2 knockout did not impact the self-renewal ability or differentiation potential into three germ layers of hESCs. Accordingly, this cell line provides a valuable model for further assessing SHMT2 functions in human embryonic development.

Changes in the circRNA expression profile of PC12 cells induced by TDCIPP exposure may regulate the downstream NF-κB pathway via the Traf2 gene.

As a commonly used organophosphorus flame retardant (OPFR), tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) has become an environmental pollutant. Studies have shown that TDCIPP exposure has many toxic effects, such as neurotoxicity, reproductive development toxicity and endocrine disturbance. CircRNAs are circular noncoding RNAs that have been found to play important roles in the occurrence of a variety of diseases. However, it is unclear whether circRNAs play a role in the cytotoxicity induced by TDCIPP exposure. In this study, we analysed the circRNA microarray results of a control group and a TDCIPP exposure group of PC12 cells and detected 3432 differentially expressed circRNAs (P < 0.05), of which 1682 were upregulated and 1750 were downregulated in the TDCIPP-exposure group. The expression levels of 2 upregulated and 3 downregulated circRNAs were verified by real-time quantitative PCR, and the results were consistent with the microarray results. Then, ceRNA analysis was performed on several kinds of circRNAs to predict the possible binding miRNAs and binding sites. The target genes were analysed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Through predictive analysis, it was found that rno_circRNA_013845, rno-miR-361-3p, and rno-miR-702-3p may be involved in the regulation of Traf2 expression, thereby affecting the expression of the downstream NF-κB signalling pathway and causing apoptosis.

USP39 regulates DNA damage response and chemo-radiation resistance by deubiquitinating and stabilizing CHK2.

The serine/threonine kinase, CHK2 (checkpoint kinase 2), is a key mediator in DNA damage response and a tumor suppressor, which is implicated in promoting cell cycle arrest, apoptosis and DNA repair. Accumulating evidence suggests that these functions are primarily exerted through phosphorylation downstream factors such as p53 and BRCA1. Recent studies have shown that ubiquitination is an important mode of regulation of CHK2. However, it remains largely unclear whether deubiquitinases participate in regulation of CHK2. Here, we report that a deubiquitinase, USP39, is a new regulator of CHK2. Mechanistically, USP39 deubiquitinates and stabilizes CHK2, which in turn enhances CHK2 stability. Short hairpin RNA (shRNA) mediated knockdown of USP39 led to deregulate CHK2, which resulted in compromising the DNA damage-induced G2/M checkpoint, decreasing apoptosis, and conferring cancer cells resistance to chemotherapy drugs and radiation treatment. Collectively, we identify USP39 as a novel regulator of CHK2 in the DNA damage response.

JNK activation mediates the apoptosis of xCT-deficient cells.

System X(c)(-) is an anionic amino acid transport system highly specific for cystine and glutamate. The underlying mechanism of cell death of cultured cells from the subtle gray (sut) mouse which contains an xCT null mutation remains elucidated. Our results show that the death of sut cells is likely caused by apoptosis mediated by c-Jun N-terminal kinase (JNK). The JNK activation triggers both a caspase-dependent (caspases-9 and -3) and an ER stress-mediated (eIF2 and CHOP) pathway to induce apoptosis. These findings suggest the possible pathways involved in the cell death of xCT-deficient cells.

xCT deficiency induces autophagy via endoplasmic reticulum stress activated p38-mitogen-activated protein kinase and mTOR in sut melanocytes.

xCT, the functional subunit of the system xc(-) encoded by the Slc7a11 gene, plays an important role in maintaining intracellular glutathione (GSH) levels. In previous study, we have indicated that xCT deficiency induces OS and that OS triggers apoptosis through JNK pathway, however, this induction of apoptotic features did not fully explain the cell death induced by xCT deficiency. In the current study, we demonstrated that sut melanocytes of xCT deficiency showed activation of both ER stress and autophagy. And that the activation of autophagy by xCT deficiency was mediated by ER stress induced activation of p38 MAPK and NF-κB pathways and subsequently inhibited functions of Akt/mTOR/p70S6K survival pathways, ultimately led to autophagic cell death of sut melanocytes. Our novel results provided important insights into understanding the mechanism associated with xCT deficiency.

Effects of tris(1,3-dichloro-2-propyl)phosphate on pathomorphology and gene/protein expression related to thyroid disruption in rats.

Previous studies demonstrated that tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) caused adverse effects on thyroid hormone (TH) imbalance in aquatic and avian organisms. This study focused on the effects of TDCIPP on thyroid function and hormone homeostasis in mammals. Pubertal female Sprague-Dawley rats were orally administered 50, 100, or 250 mg per kg per d of TDCIPP from postnatal day (PND) 22 to PND42 for 21 days. The serum triiodothyronine (T3) levels increased significantly at 250 mg per kg per d of TDCIPP. There were no significant differences in the body weight, serum thyroxine (T4) and free thyroxine (FT4) levels between the control and TDCIPP treated groups. There were significant dose-dependent increases in the mRNA and protein expression levels of genes related to drug metabolism (cytochrome-p450-3A1, CYP3A1) and TH clearance (udp-glucuronosyltransferase-1A6, UGT1A6) in the liver. Treatment with TDCIPP increased hepatic type 1 deiodinase (DIO1) mRNA at 250 mg per kg per d but down-regulated hepatic TH receptor beta (TRß) mRNA expression. In addition, TDCIPP exposure induced slight thyroid follicular hyperplasia, and several genes involved in TH biosynthesis (NIS, TPO, Tg) were altered at 100 and 250 mg per kg per d of TDCIPP. Nevertheless, serum thyroid stimulating hormone (TSH) levels and the receptor (TSHr) mRNA significantly decreased at only the low dose group. Based on these results, we certified that TDCIPP disturbs the normal bioprocess on TH synthesis, biotransformation or clearance, and hepatic detoxification of pubertal female SD rats, causing thyroid function disorder.

Comparative allozyme analysis of severel grasshopper species.

The allele frequency of four allozyme loci in four grasshopper species from two families of Catantopidae and Oedipodidae was examined using horizontal starch gel electrophoresis. The zymograms show that all four species have two loci in malate dehydrogenase (MDH). At MDH-1 one moderately migrated allele is shared and dominant in all four species. Locusta migratoria manilensi has two allele fixations in lactate dehydrogenase (LDH) and malic enzyme (ME); while Gastrimargus saussure has a fixed allele at MDH-1 and a unique fixation at MDH-2. The overall genetic variance was the highest in Oxya chinensis (Allele per locus = 3.0; He = 0.22), but the lowest in L. migratoria manilens (Allele per locus = 1.5; He = 0.013). Allozyme data suggests the four species are close in phylogenetic relationship, but differentiated in genetic variation levels.

Functional protection of learning and memory abilities in rats with vascular dementia.

PURPOSE: The present study clarified the effects of repetitive transcranial magnetic stimulation (rTMS) in rats with vascular dementia (VaD) and explored the underlying mechanisms. METHODS: Two-vessel occlusion was used as a VaD model. Two weeks after carotid artery occlusion, high (5 Hz) or low (1 Hz) frequency rTMS were applied for 10 days. Spatial learning and memory abilities were tested with a Morris water maze. Hippocampal CA1 neurons were histologically examined. The expressions of mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E (eIF-4E) in CA1 were detected by western blot, and immunohistochemistry. RESULTS: Unlike unlesioned control animals, VaD rats had an impaired morphology of CA1 neurons and a reduced ability of spatial memory. rTMS significantly improved both, the morphology and the learning and memory abilities of VaD rats compared to untreated lesioned rats. Protein expressions of mTOR and eIF-4E in CA1 of VaD rats were lower than in control rats but rTMS significantly increased the expression compared to untreated VaD rats. CONCLUSIONS: rTMS promotes recovery of learning and memory abilities of VaD rats. Molecular analysis suggests that the beneficial effect of rTMS may be partly induced by upregulation of protein expressions of mTOR and eIF-4E in CA1.

Proteomic Investigation of Changes in Rat Skeletal Muscle after Exercise-Induced Fatigue

The mechanisms of exercise-induced fatigue have not been investigated using proteomic techniques, an approach that could improve our understanding and generate novel information regarding the effects of exercise. In this study, the proteom alterations of rat skeletal muscle were investigated during exercise-induced fatigue. The proteins were extracted from the skeletal muscle of SD rat thigh, and then analyzed by two-dimensional electrophoresis and PDQuest software. Compared to control samples, 10 significantly altered proteins were found in exercise samples, two of them were upregulated and eight of them were downregulated. These proteins were identified by MALDI TOF-MS. The two upregulated proteins were identified as MLC1 and myosin L2 (DTNB) regulatory light-chain precursors. The eight decreased proteins are Glyceraldehyde-3-phosphate Dehydrogenas (GAPDH); Beta enolase; Creatine kinase M chain (M-CK); ATP-AMP Transphosphorylase (AK1); myosin heavy chain (MHC); actin; Troponin I, fast-skeletal muscle (Troponin I fast-twitch isoform), fsTnI; Troponin T, fast-skeletal muscle isoforms (TnTF). In these proteins, four of the eight decreased proteins are related directly or indirectly to exercise induced fatigue. The other proteins represent diverse sets of proteins including enzymyes related to energy metabolism, skeletal muscle fabric protein and protein with unknown functions. They did not exhibit evident relationship with exercise-induced fatigue. Whereas the two identified increased proteins exhibit evident relationship with fatigue. These findings will help in understanding the mechanisms involved in exercise-induced fatigue.