L-Carnitine protects mammalian cells from chromosome aberrations but not from inhibition of cell proliferation induced by hydrogen peroxide

L-carnitine is a small essential molecule indispensable in fatty acid metabolism and required in several biological pathways regulating cellular homeostasis. Despite considerable progress in understanding of L-carnitine biosynthesis and metabolism, very few data are reported concerning the protective role of L-carnitine from oxidative stress-induced DNA damage that is known to be a factor in cell transformation and tumourigenesis. In order to detect the capability of L-carnitine to protect mammalian cells from oxidative stress-induced chromosomal effects, we analysed chromosome aberrations in mitotic CHO cells, which represent an appropriate cytogenetic model to study compounds that enhance cell protection against externally induced DNA damage. We chose H2O2 as an inducer of oxidative stress. Our results demonstrate for the first time a marked and reproducible reduction of H2O2-induced chromosome damage involving an L-carnitine-mediated capacity to buffer intracellular formation of reactive oxygen species (ROS). Furthermore, by studying the mitotic index and cell cycle progression, we also demonstrated that this protective effect is highly specific, since L-carnitine itself was not able to prevent the inhibition of cell growth caused by H2O2.     

Elasticity theory of the B-DNA to S-DNA transition.

We propose in this note a simple model--the two-state Worm Like Chain--to describe the elasticity of the recently discovered stress-induced transformation from B-DNA to S-DNA. The model reduces for low tractions to the well-known Worm Like chain theory, which is used to describe the elastic properties of B-DNA, while in the limit of high chain-bending moduli it reduces to the two-state Ising model proposed by Cluzel et al. for the B-S transition [Cluzel, P., A. Lebrun, C. Heller, R. Lavery, J-L. Viovy, D. Chatenay, and F. Caron. 1996. DNA: an extensible molecule. Science. 271:792-794]. Our model can be treated analytically to produce an explicit form of the force-extension relationship which agrees reasonably with the observations. We use the model to show that conformational fluctuations of the chain play a role also for the B to S transformation.     

Transformation of physiological gastroprotective effects of glucocorticoids into pathological ulcerogenic consequences

The study was designed to investigate how physiological gastroprotective action of glucocorticoids could be transformed to pathological proulcerogenic effect. Time-dependent effects of single injection of dexamethasone on stress-induced gastric erosions, corticosterone and blood glucose levels, somatic parameters were investigated in fasted rats. Dexamethasone injected at the same dose attenuated or aggravated the stress-induced gastric erosions depending on the time of the injection. In case of dexamethasone injection 1-12 hrs before stress, we observed its gastroprotective action. Further increase in the time interval caused transformation of the gastroprotective action of dexamethasone to proulcerogenic effect. Accordingly to the results obtained, dexamethasone-induced long-lasting maintenance of blood glucose levels accompanied with signs of catabolic effect as well as dexamethasone-induced corticosterone deficiency may be responsible, at least partly, for the transformation of gastroprotective effect of dexamethasone to the proulcerogenic one.     

Global and local depletion of ternary complex limits translational elongation

The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms.     

AIP1 is critical in transducing IRE1-mediated endoplasmic reticulum stress response

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.     

Quinotrierixin Inhibited ER Stress-Induced XBP1 mRNA Splicing through Inhibition of Protein Synthesis

Quinotrierixin was isolated from microbes as an inhibitor of ER stress-induced XBP1 mRNA splicing, but its mode of action was unclear. We found that quinotrierixin is an inhibitor of protein synthesis, and that the required dose range of quinotrierixin to inhibit ER stress-induced XBP1 mRNA splicing was similar to that to inhibit protein synthesis. Furthermore, we also found that quinotrierixin inhibited the ER stress-induced increases of unfolded protein response-related genes such as GRP78, CHOP, EDEM, ERdj4, and p58^IPK. Thus, we showed that quinotrierixin inhibited the ER stress-induced unfolded protein response, possibly due to its inhibitory activity of protein synthesis.     

Quinotrierixin inhibited ER stress-induced XBP1 mRNA splicing through inhibition of protein synthesis

Quinotrierixin was isolated from microbes as an inhibitor of ER stress-induced XBP1 mRNA splicing, but its mode of action was unclear. We found that quinotrierixin is an inhibitor of protein synthesis, and that the required dose range of quinotrierixin to inhibit ER stress-induced XBP1 mRNA splicing was similar to that to inhibit protein synthesis. Furthermore, we also found that quinotrierixin inhibited the ER stress-induced increases of unfolded protein response-related genes such as GRP78, CHOP, EDEM, ERdj4, and p58(IPK). Thus, we showed that quinotrierixin inhibited the ER stress-induced unfolded protein response, possibly due to its inhibitory activity of protein synthesis.     

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G₂/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.     

细胞衰老与细胞自噬的生物学关联及其意义

细胞衰老是指细胞生理功能的衰减,包括增殖能力下降、细胞周期停滞、对促凋亡应激不敏感、衰老相关基因和蛋白表达增加,伴有形态学衰老改变,渐趋死亡的现象,其至少可分为复制性衰老和应激诱导的衰老。细胞自噬属于细胞＂自食＂现象,是细胞依赖溶酶体的分解代谢过程,能降解受损蛋白、衰老或损伤的细胞器等细胞结构,可被多种应激所触发。细胞自噬的典型特征是形成自噬体并呈递给溶酶体,该过程在蛋白质和细胞器质量控制中起基础作用并维持了细胞能量的稳态。最新研究表明,自噬与细胞衰老密切相关,参与蛋白酶和自噬相关调节的BAG蛋白家族中BAG3/BAG1比值在复制性衰老时增高,且BAG3在细胞衰老时能介导自噬的激活。在Ras诱导的细胞衰老进程中亦可观察到较高的自噬活性。再者,自噬作为生物机体抗衰老的效应因子的遗传学证据已在低等真核生物中发现。还有研究证实,作为人类精液主要组分的亚精胺能够触发组蛋白H3脱乙酰基作用,此改变上调了自噬相关转录物的表达,继而引发自噬活性增强,从而延缓了多种细胞的衰老进程。另有研究显示,在P53/Arf的正常调节下,小鼠的衰老进程得以延缓,而Arf在细胞自噬过程的调节中亦是不可或缺的。总之,自噬活性的改变影响细胞衰老进程并可作为细胞衰老新的效应机理。     

Expression of stress-evoked analgesia, connected with activity of animals in a forced swimming test]

The influence of forced swimming on the development of stress-induced analgesia was studied in 35 SHR mice, 65 NMRI mice, and 23 white outbred male rats. Mice were subjected to swimming conditions (at a temperature of 11 degrees C) for a period of 4 minutes and rats for 6 minutes. Pain thresholds were measured by a footshock. It was shown that behavioral response to acute stress is associated with a change in the pain tolerance threshold: activity of an animal under test conditions positively correlated with stress-induced analgesia. The response to stress and parameters of stress-induced analgesia depend on the genetic factor and age, however, the correlation between the activity during exposure to stress and the extent of stress-induced analgesia conserves in all cases.     

Mutability and importance of a hypermutable cell subpopulation that produces stress-induced mutants in Escherichia coli

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac⁺ mutants, with and without evidence of descent from the HMS, have similar Lac⁺ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac⁺ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.     

Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H₂O₂-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H₂O₂ treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.     

The proteomic to biology inference,a frequently overlooked concern in the interpretation of proteomic data: A plea for functional validation

Proteomics will celebrate its 20th year in 2014. In this relatively short period of time, it has invaded most areas of biology and its use will probably continue to spread in the future. These two decades have seen a considerable increase in the speed and sensitivity of protein identification and characterization, even from complex samples. Indeed, what was a challenge twenty years ago is now little more than a daily routine. Although not completely over, the technological challenge now makes room to another challenge, which is the best possible appraisal and exploitation of proteomic data to draw the best possible conclusions from a biological point of view. The point developed in this paper is that proteomic data are almost always fragmentary. This means in turn that although better than an mRNA level, a protein level is often insufficient to draw a valid conclusion from a biological point of view, especially in a world where PTMs play such an important role. This means in turn that transformation of proteomic data into biological data requires an important intermediate layer of functional validation, i.e. not merely the confirmation of protein abundance changes by other methods, but a functional appraisal of the biological consequences of the protein level changes highlighted by the proteomic screens.     

Dietary Fish Oil Inhibits Pro-Inflammatory and ER Stress Signalling Pathways in the Liver of Sows during Lactation

Lactating sows have been shown to develop typical signs of an inflammatory condition in the liver during the transition from pregnancy to lactation. Hepatic inflammation is considered critical due to the induction of an acute phase response and the activation of stress signaling pathways like the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), both of which impair animal´s health and performance. Whether ER stress-induced UPR is also activated in the liver of lactating sows and whether dietary fish oil as a source of anti-inflammatory effects n-3 PUFA is able to attenuate hepatic inflammation and ER stress-induced UPR in the liver of sows is currently unknown. Based on this, two experiments with lactating sows were performed. The first experiment revealed that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a set of genes regulated by the UPR and numerically increased phosphorylation of the ER stress-transducer PERK and PERK-mediated phosphorylation of eIF2α and IκB. The second experiment showed that fish oil inhibits ER stress-induced UPR in the liver of lactating sows. This was demonstrated by decreased mRNA levels of a number of UPR-regulated genes and reduced phosphorylation of PERK and PERK-mediated phosphorylation of eIF2α and IκB in the liver of the fish oil group. The mRNA levels of various nuclear factor-κB-regulated genes encoding inflammatory mediators and acute phase proteins in the liver of lactating sows were also reduced in the fish oil group. In line with this, the plasma levels of acute phase proteins were reduced in the fish oil group, although differences to the control group were not significant. In conclusion, ER stress-induced UPR is present in the liver of lactating sows and fish oil is able to inhibit inflammatory signaling pathways and ER stress-induced UPR in the liver.     

Transmembrane Protein 214 (TMEM214) Mediates Endoplasmic Reticulum Stress-induced Caspase 4 Enzyme Activation and Apoptosis

Endoplasmic reticulum (ER) stress caused by excessive aggregation of misfolded proteins induces apoptosis. Although ER stress-induced apoptosis has been implicated in many diseases, the detailed mechanisms are not well understood. Here, we identified human transmembrane protein 214 (TMEM214) as a critical mediator of ER stress-induced apoptosis. Overexpression of TMEM214 induced apoptosis, whereas knockdown of TMEM214 inhibited ER stress-induced apoptosis. TMEM214 was localized on the outer membrane of the ER and constitutively associated with procaspase 4, which was also critical for ER stress-induced apoptosis. TMEM214-induced apoptosis was abolished by a dominant negative mutant of procaspase 4, whereas caspase 4-induced apoptosis was inhibited by knockdown of TMEM214. Furthermore, knockdown of TMEM214 inhibited the activation and cleavage of procaspase 4 by impairing its recruitment to the ER. Our findings suggest that TMEM214 is essential for ER stress-induced apoptosis by acting as an anchor for recruitment of procaspase 4 to the ER and its subsequent activation.