Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. Here we identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized alpha-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.     

Caspase-3在roscovitine诱发PC12细胞凋亡中发挥重要作用

我们已证实周期蛋白激酶（cyclin-dependent kinases）cdk2、cdc2和cdk5抑制剂roscovitine诱导PC12细胞凋亡。本实验应用caspase-3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法,研究了caspase-3在roscovitine所致PC12细胞凋亡中的作用。结果显示,roscovitine（50μmol／L）处理PC12细胞12h,细胞核染色质凝缩及核碎片形成,同时胞浆中出现caspase-3阳性标志,caspase-3阳性细胞占细胞总数的42％。非特异性caspases抑制剂Z-VAD-FMK（50μmol／L）和caspase-3特异性抑制剂Z-DEVD-FMK（100μmol／L）可部分降低roscovitine所致的细胞死亡,使细胞存活率分别由29．03％（roscovitine）增至58．06％（Z-VAD-FMK＋roscovitine）和45．16％（Z-DEVD-FMK＋roscovitine）：用单克隆non-erythroid α-spectrin抗体检测roscovitine处理组细胞匀浆提取液,表明caspase-3裂解的特异性spectfin 120kDa蛋白产物较对照组显著增加。提示细胞凋亡成分caspases参与roscovitine所敛的细胞凋亡,其中caspase-3发挥重要作用。     

Epigenetic cell reactions induced by ionizing radiation

The importance of radiation modification of epigenetic activity in the general mechanism of radiobiological reactions is proved. The inheritable epigenetic changes induced by irradiation are one of the basic reasons of formation of the remote radiation pathology. It is noted that epigenetic inheritable changes of cells have the determined character distinguishing them from mutation changes, being individual and not directed. It is underlined the ability of ionizing radiation to modify a level of spontaneous genetic instability inherited in a number of cell generations on the epigenetic mechanism.     

Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.     

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.     

Bacterial virulence plays a crucial role in MRSA sepsis

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

Mutational alterations induced in yeast by ionizing radiation

The cyc1-9 ochre (UAA) mutant and the cyc1-179 amber (UAG) mutant of the yeast Saccharomyces cerevisiae were reverted with X-rays and -particles. The amino acid sequence changes of iso-1-cytochromes c from 36 of the intragenic revertants were determined by amino acid analysis and peptide mapping, aided by partial amino acid sequencing of 4 revertants. In addition, the DNA segments encompassing 3 unusual mutations with complex changes were cloned and sequenced. This study and previous studies of 16 other revertants of cyc1-9 and cyc1-179 revealed that ionizing radiation primarily induces single base-pair substitutions; 47 of the 52 revertants arose by transversions and transitions without any apparent preference. However, the A·T→T·A substitution at the first base pair for the cyc1-179 UAG codon, leading to the normal protein, was not detected, nor was it found previously in 32 revertants of cycl-179 obtained spontaneously or induced with various other mutagens; apparently, there is a prohibition of certain base-pair substitutions at certain sites in DNA. In addition, 5 of the 52 revertants arose by multiple changes within a short region of 11 base pairs. These consisted of the deletion of 6 base pairs, the substitution of 3 base pairs, and 3 different kinds of substitutions of two base pairs. Compared to other mutagens previously tested with the cyc1 system, ionizing radiation produces the most random types of base-pair substitutions.     

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.     

NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a collection of diseases in which rod photoreceptors die from a variety of mutations. After rods die, the level of tissue oxygen in the outer retina becomes elevated and there is progressive oxidative damage to cones that ultimately triggers apoptosis. In this study, we investigated the hypothesis that NADPH oxidase (Nox) and/or xanthine oxidase serve as critical intermediaries between increased tissue oxygen and the generation of excessive reactive oxygen species that cause oxidative damage to cones. Apocynin, a blocker of Nox, but not allopurinol, a blocker of xanthine oxidase, markedly reduced the superoxide radicals visualized by hydroethidine in the outer retina in the retinal degeneration-1 ( rd1 ^+/+ ) model of RP. Compared to rd1 ^+/+ mice treated with vehicle, those treated with apocynin, but not those treated with allopurinol, had significantly less oxidative damage in the retina measured by ELISA for carbonyl adducts. Apocynin-treated, but not allopurinol-treated, rd1 ^+/+ mice had preservation of cone cell density, increased mRNA levels for m- and s-cone opsin, and increased mean photopic b-wave amplitude. In Q344ter mice, a model of dominant RP in which mutant rhodopsin is expressed, apocynin treatment preserved photopic electroretinogram b-wave amplitude compared to vehicle-treated controls. These data indicate that Nox, but not xanthine oxidase, plays a critical role in generation of the oxidative stress that leads to cone cell death in RP and inhibition of Nox provides a new treatment strategy.     

MTBP plays a crucial role in mitotic progression and chromosome segregation

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint.     

The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

iNOS in cardiac myocytes plays a critical role in death in a murine model of hypertrophy induced by calcineurin

Transgenic overexpression of calcineurin (CN/Tg) in mouse cardiac myocytes results in hypertrophy followed by dilation, dysfunction, and sudden death. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in cardiac injury. Since calcineurin regulates iNOS expression, and since phenotypes of mice overexpressing iNOS are similar to CN/Tg, we hypothesized that iNOS is pathogenically involved in cardiac phenotypes of CN/Tg mice. CN/Tg mice had increased serum and cardiac iNOS levels. When CN/Tg-iNOS(-/-) and CN/Tg mice were compared, some phenotypes were similar: extent of hypertrophy and fibrosis. However, CN/Tg-iNOS(-/-) mice had improved systolic performance (P < 0.001) and less heart block (P < 0.0001); larger sodium current density and lower serum TNF-alpha levels (P < 0.03); and less apoptosis (P < 0.01) resulting in improved survival (P < 0.0003). To define tissue origins of iNOS production, chimeric lines were generated. Bone marrow (BM) from wild-type or iNOS(-/-) mice was transplanted into CN/Tg mice. iNOS deficiency restricted to BM-derived cells was not protective. Calcineurin activates the local production of NO by iNOS in cardiac myocytes, which significantly contributes to sudden death, heart block, left ventricular dilation, and impaired systolic performance in this murine model of cardiac hypertrophy induced by the overexpression of calcineurin.     

IEC-6 intestinal cell death induced by bovine milk alpha-lactalbumin

Potent inhibition of cell proliferation was found for commercial preparations of bovine alpha-lactalbumin on cultured intestinal cell line IEC-6 albeit lot-dependent. The inhibition was irreversible and a single exposure to the culture medium containing alpha-lactalbumin of an active lot for a period as short as 30 min was enough to provoke cell death, possibly through apoptosis. The oligomer fraction from size exclusion chromatography was significantly robust, while the monomer fraction remained totally inert, in inducing cell death. Incubation at 37 degrees C for 5 d with 30% trifluoroethanol in acetate, pH 5.5, in a slowly rotating test tube rendered the monomer fraction cytotoxic. Again, the resulting inhibitory activity was found in the oligomer fraction from size exclusion chromatography, with emergence of subtle peaks at 22- and 30-kDa. Furthermore, the occurrence of SDS-stable 30-kDa as well as 20-kDa bands on electrophoresis was a common feature for alpha-lactalbumin with the activity inducing cell death. Thus, a certain dimeric state can be implicated in the cytotoxicity of bovine alpha-lactalbumin.