T-2 toxin is a worldwide trichothecenetoxin and can cause various toxicities.T-2 toxin is involved in G1 phase arrest in several cell lines but molecular mechanism is still not clear. In present study, we used rat pituitary GH3 cells to investigate the mechanism involved in cell cycle arrest against T-2 toxin (40 nM) for 12, 24, 36 and 48 h as compared to control cells. GH3 cells showed a considerable increase in reactive oxygen species (ROS) as well as loss in mitochondrial membrane potential (△Ym) upon exposure to the T-2 toxin. Flow cytometry showed a significant time-dependent increase in percentage of apoptotic cells and gel electrophoresis showed the hallmark of apoptosis oligonucleosomal DNA fragmentation. Additionally, T-2 toxin-induced oxidative stress and DNA damage with a time-dependent significant increased expression of p53 favors the apoptotic process by the activation of caspase-3 in T-2 toxin treated cells. Cell cycle analysis by flow cytometry revealed a time-dependent increase ofG1 cell population along with the significant time-dependent up-regulation of mRNA and protein expression of p16 and p21 and significant down-regulation of cyclin D1, CDK4, and p-RB levels further verify the G1 phase arrest in GH3 cells. Morphology of GH3 cells by TEM clearly showed the damage and dysfunction to mitochondria and the cell nucleus. These findings for the first time demonstrate that T-2 toxin induces G1 phase cell cycle arrest by the involvement of p16/Rb pathway, along with ROS mediated oxidative stress and DNA damage with p53 and caspase cascade interaction, resulting in apoptosis in GH3 cells.

• MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   fyziologie   MeSH
• geny p16 účinky léků   fyziologie   MeSH
• hypofýza účinky léků   metabolismus   ultrastruktura   MeSH
• krysa rodu rattus MeSH
• retinoblastomový protein biosyntéza   MeSH
• signální transdukce účinky léků   fyziologie   MeSH
• T-2 toxin toxicita   MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chronic exposure to trichothecenes is known to disturb insulin-like growth factor 1 and signaling of insulin and leptin hormones and causes considerable growth retardation in animals. However, limited information was available on mechanisms underlying trichothecene-induced growth retardation. In this study, we employed an integrated transcriptomics, proteomics, and RNA interference (RNAi) approach to study the molecular mechanisms underlying trichothecene cytotoxicity in rat pituitary adenoma GH3 cells. Our results showed that trichothecenes suppressed the synthesis of growth hormone 1 (Gh1) and inhibited the eukaryotic transcription and translation initiation by suppressing aminoacyl-tRNA synthetases transcription, inducing eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) and reducing eukaryotic translation initiation factor 5 a. The sulfhydryl oxidases , protein disulfide isomerase,and heat shock protein 90 (were greatly reduced, which resulted in adverse regulation of protein processing and folding. Differential genes and proteins associated with a decline in energy metabolism and cell cycle arrest were also found in our study. However, use of RNAi to interfere with hemopoietic cell kinase (Hck) and EIF2AK2 transcriptions or use of chemical inhibitors of MAPK, p38, Ras, and JNK partially reversed the reduction of Gh1 levels induced by trichothecenes. It indicated that the activation of MAPKs, Hck, and EIF2AK2 were important for trichothecene-induced growth hormone suppression. Considering the potential hazards of exposure to trichothecenes, our findings could help to improve our understanding regarding human and animal health implications.

• MeSH
• aminoacyl-tRNA-synthetasy antagonisté a inhibitory   MeSH
• apoptóza účinky léků   MeSH
• buněčné linie MeSH
• genetická transkripce účinky léků   MeSH
• iniciační faktory antagonisté a inhibitory   MeSH
• kinasa eIF-2 antagonisté a inhibitory   MeSH
• krysa rodu rattus MeSH
• oxidoreduktasy antagonisté a inhibitory   MeSH
• proteindisulfidisomerasy antagonisté a inhibitory   MeSH
• proteiny tepelného šoku HSP90 antagonisté a inhibitory   MeSH
• proteiny vázající RNA antagonisté a inhibitory   MeSH
• proteomika * MeSH
• proteosyntéza účinky léků   MeSH
• RNA interference účinky léků   MeSH
• růstový hormon antagonisté a inhibitory   MeSH
• stanovení celkové genové exprese * MeSH
• T-2 toxin analogy a deriváty   farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chronic exposure to low dose of T-2 toxin causes growth retardation and reduced body weight, resulting in economic losses. Excessive inflammatory cytokines and GH deficiency are important mechanisms that contribute to growth inhibition induced by T-2 toxin. However, the regulation of the inflammatory cytokines expecially IL-6, IL-1β, and TNF-α induced by T-2 toxin still remained unclear. The new transcription factor AKNA, belonging to AT-hook protein family, is closely associated with inflammation. However, it was unclear how AKNA regulate the expression of inflammatory cytokines, and there was no report on the role of AKNA in T-2 toxin mediated toxicity. Here, we investigated the role of AKNA in T-2 toxin-mediated inflammatory response and GH deficiency and the signal transduction pathway of AKNA. We showed that AKNA regulated by PKA/CREB and NF-κB pathway is a novel downstream molecular target in T-2 toxin-mediated inflammation and GH deficiency. T-2 toxin activates the PKA/CREB and NF-κB/p65 pathways, thereby promoting the direct binding of phospho-CREB and phospho-p65 to the AKNA promoter, thus inhibiting AKNA expression. GH and inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression were significantly downregulated after AKNA silencing. Furthermore, the expression of differential genes induced by T-2 toxin in the rat pituitary was further confirmed by acute toxicity tests in rats, which was consistent with the results in GH3 cells. By histopathological analysis, we confirmed the pituitary might be a novel direct target organ of T-2 toxin. These findings provided new insights into the significant role of AKNA in T-2 toxin-induced inflammatory response and growth inhibition.

• MeSH
• buněčné linie MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• down regulace MeSH
• fosforylace MeSH
• interleukin-1beta genetika   metabolismus   MeSH
• interleukin-6 genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• NF-kappa B metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• protein vázající element responzivní pro cyklický AMP metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• T-2 toxin toxicita   MeSH
• TNF-alfa genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• umlčování genů MeSH
• zánět chemicky indukované   genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The present study was conducted to determine the gene responsible for beta-glucosidase (BGL) production and to generate a full-length complementary DNA (cDNA) of one of the putative BGL genes, which showed a significant expression level when Schizophyllum commune KUC9397 was grown in optimized medium. The relative expression levels of seven genes encoding BGL of S. commune KUC9397 were determined with real-time quantitative reverse transcription PCR in cellulose-containing optimized medium (OM) compared to glucose-containing basal medium (BM). The most abundant transcript was bgl3a in OM. The transcript number of the bgl3a increased more than 57.60-fold when S. commune KUC9397 was grown on cellulose-containing OM compared to that on glucose-containing BM. The bgl3a was identified, and a deduced amino acid sequence of bgl3a shared homology (97%) with GH3 BGL of S. commune H4-8. This is the first report showing the transcription levels of genes encoding BGL and identification of full-length cDNA of glycoside hydrolase 3 (GH3) BGL from S. commune. Furthermore, this study is one of the steps for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

• MeSH
• aktivace transkripce MeSH
• beta-glukosidasa biosyntéza   genetika   MeSH
• kultivační média chemie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• molekulární sekvence - údaje MeSH
• Schizophyllum enzymologie   genetika   růst a vývoj   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• stanovení celkové genové exprese * MeSH
• upregulace MeSH
• Publikační typ
• časopisecké články MeSH

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• biologické modely MeSH
• dioxygenasy metabolismus   MeSH
• fenotyp MeSH
• fylogeneze MeSH
• homeostáza * MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• metabolomika MeSH
• mutace genetika   MeSH
• oxidace-redukce MeSH
• promotorové oblasti (genetika) genetika   MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny * MeSH
• sekvence aminokyselin MeSH
• semenáček metabolismus   MeSH
• výhonky rostlin metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-β-glucosidase I and II (αRβG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-β-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRβG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRβG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRβG I. On the other hand, αRβG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRβG I showed exclusive specificity toward 7-O-β-rutinosyl flavonoids, whereas αRβG II hydrolyzed both 7-O-β-rutinosyl- and 3-O-β-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-β-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRβG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

• MeSH
• Acremonium enzymologie   genetika   MeSH
• flavonoidy metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• fylogeneze MeSH
• glykosidhydrolasy genetika   metabolismus   MeSH
• molekulová hmotnost MeSH
• Pichia genetika   MeSH
• rekombinantní proteiny metabolismus   MeSH
• sekvenční analýza DNA MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH

Deoxynivalenol (DON) is an unavoidable contaminant in human food, animal feeds, and agricultural products. Growth retardation in children caused by extensive DON pollution has become a global problem that cannot be ignored. Previous studies have shown that DON causes stunting in children through intestinal dysfunction, insulin-like growth factor-1 (IGF-1) axis disorder and peptide YY (PYY). Galanin-like peptide (GALP) is an important growth regulator, but its role in DON-induced growth retardation is unclear. In this study, we report the important role of GALP during DON-induced growth inhibition in the rat pituitary tumour cell line GH3. DON was found to increase the expression of GALP through hypomethylationin the promoter region of the GALP gene and upregulate the expression of proinflammatory factors, while downregulate the expression of growth hormone (GH). Furthermore, GALP overexpression promoted proinflammatory cytokines, including TNF-α, IL-1β, IL-11 and IL-6, and further reduced cell viability and cell proliferation, while the inhibitory effect of GALP was the opposite. The expression of GALP and insulin like growth factor binding protein acid labile subunit (IGFALS) showed the opposite trend, which was the potential reason for the regulation of cell proliferation by GALP. In addition, GALP has anti-apoptotic effects, which could not eliminate the inflammatory damage of cells, thus aggravating cell growth inhibition. The present findings provide new mechanistic insights into the toxicity of DON-induced growth retardation and suggest a therapeutic potential of GALP in DON-related diseases.

• MeSH
• apoptóza MeSH
• epigeneze genetická účinky léků   MeSH
• galanin genetika   metabolismus   MeSH
• glykoproteiny genetika   metabolismus   MeSH
• hypofýza cytologie   MeSH
• krysa rodu rattus MeSH
• nádorové buněčné linie MeSH
• proliferace buněk MeSH
• transportní proteiny genetika   metabolismus   MeSH
• trichotheceny farmakologie   MeSH
• umlčování genů MeSH
• upregulace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH