Heat-shock proteins maintain the viability of ATP-deprived cells: what is the mechanism?

ATP depletion causes necrosis in mammalian cells. However, a previous heat shock can protect cells from the effects of energy deprivation, probably as a result of the synthesis and accumulation of heat-shock proteins (hsps). We propose that hsps protect ATP-depleted cells from rapid necrotic death by inhibiting the aggregation of cytoskeletal proteins that occurs when ATP synthesis is blocked.     

Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

Background  

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.     

Necrosis: a specific form of programmed cell death?

For a long time necrosis was considered as an alternative to programmed cell death, apoptosis. Indeed, necrosis has distinct morphological features and it is accompanied by rapid permeabilization of plasma membrane. However, recent data indicate that, in contrast to necrosis caused by very extreme conditions, there are many examples when this form of cell death may be a normal physiological and regulated (programmed) event. Various stimuli (e.g., cytokines, ischemia, heat, irradiation, pathogens) can cause both apoptosis and necrosis in the same cell population. Furthermore, signaling pathways, such as death receptors, kinase cascades, and mitochondria, participate in both processes, and by modulating these pathways, it is possible to switch between apoptosis and necrosis. Moreover, antiapoptotic mechanisms (e.g., Bcl-2/Bcl-x proteins, heat shock proteins) are equally effective in protection against apoptosis and necrosis. Therefore, necrosis, along with apoptosis, appears to be a specific form of execution phase of programmed cell death, and there are several examples of necrosis during embryogenesis, a normal tissue renewal, and immune response. However, the consequences of necrotic and apoptotic cell death for a whole organism are quite different. In the case of necrosis, cytosolic constituents that spill into extracellular space through damaged plasma membrane may provoke inflammatory response; during apoptosis these products are safely isolated by membranes and then are consumed by macrophages. The inflammatory response caused by necrosis, however, may have obvious adaptive significance (i.e., emergence of a strong immune response) under some pathological conditions (such as cancer and infection). On the other hand, disturbance of a fine balance between necrosis and apoptosis may be a key element in development of some diseases.     

HSP72 can protect cells from heat-induced apoptosis by accelerating the inactivation of stress kinase JNK

The major heat shock protein Hsp72 prevents heat-induced apoptosis. We have previously demonstrated that transiently expressed Hsp72 exerts its anti-apoptotic effect by suppressing the activity of stress-kinase JNK, an early component of the apoptotic pathway initiated by heat shock. On the other hand, constitutive expression of Hsp72 does not lead to suppression of heat-induced JNK activation, yet still efficiently prevents apoptosis. To address this apparent contradiction, we studied the effects of constitutively expressed Hsp72 on activation of JNK and apoptosis in Rat-1 fibroblasts. We found that the level of heat-induced apoptosis directly correlated with the duration rather than the magnitude of JNK activity following heat shock. Constitutively expressed Hsp72 strongly reduced the duration of JNK while it did not suppress initial JNK activation. These effects were due to Hsp72-mediated acceleration of JNK dephosphorylation. Addition of vanadate to inhibit JNK phosphatase activity completely prevented the anti-apoptotic action of Hsp72. Therefore, suppression of heat-induced apoptosis by Hsp72 could be fully accounted for by its effects on JNK activity.     

Suppression of stress kinase JNK is involved in HSP72-mediated protection of myogenic cells from transient energy deprivation. HSP72 alleviates the stewss-induced inhibition of JNK dephosphorylation

Since protection of cells from stress-induced apoptosis by the heat shock protein Hsp72 involves suppression of stress kinase JNK, we suggested that Hsp72-mediated JNK inhibition might also be critical for myocardial protection from ischemia/reperfusion. Transient energy deprivation of H9c2 myogenic cells, used as an in vitro model of myocardial ischemia, led to cell death that had morphological features of apoptosis and necrosis and was independent of caspases. Surprisingly, this unusual type of cell death was regulated by JNK and ERK kinases. In fact, specific inhibition of JNK increased cell survival; specific inhibition of ERKs enhanced deleterious consequences of energy deprivation, whereas inhibition of p38 kinase had no effect. Hsp72 suppressed activation of JNK and did not increase ERK activity, suggesting that inhibition of JNK is the important component of Hsp72-mediated protection. Upon transient energy deprivation, activation of JNK proceeds via two distinct pathways, stimulation of JNK phosphorylation by a protein kinase SEK1 and inhibition of JNK dephosphorylation. Remarkably, in cells exposed to transient energy deprivation, Hsp72 enhanced the rate of JNK dephosphorylation but did not affect SEK1 activity. Therefore, it appears that Hsp72 specifically down-regulates JNK by accelerating its dephosphorylation, which reduces the susceptibility of cardiac cells to simulated ischemia/reperfusion.     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains

Background

Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers.

Results

The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping.We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population.

Conclusion

The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under “GNU GPL v3”. The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0475-6) contains supplementary material, which is available to authorized users.     

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.     

The function of HSP72 in suppression of c-Jun N-terminal kinase activation can be dissociated from its role in prevention of protein damage.

Activation of the c-Jun N-terminal kinase (JNK) by a variety of stimuli is critical for regulation of many cellular processes including apoptosis. The major inducible heat shock protein Hsp72 has previously been demonstrated to inhibit activation of JNK in cells exposed to heat shock and other protein-damaging agents, thus suppressing apoptosis. Hsp72 can protect proteins from stress-induced damage. To test if this protective function of Hsp72 is involved in JNK suppression, we investigated whether Hsp72 can avert activation of JNK by stimuli that do not cause protein damage. We show that Hsp72 suppresses activation of JNK induced by non-protein-damaging stimuli, interleukin-1 and UV irradiation, as well as by constitutively active components of the JNK signaling cascade Cdc42 and MEKK1. Furthermore, Hsp72 strongly reduced activation of JNK by phosphatase inhibitors. We also demonstrate that an Hsp72 mutant that lacks the ATPase domain is still capable of JNK suppression, thus indicating that the protein refolding activity of Hsp72 is not critical for inhibition of JNK activation. Taken together these data suggest that Hsp72 plays a regulatory role in JNK signaling and that the function of Hsp72 in protein protection or refolding is not involved in JNK regulation.