The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells

The Notch signaling pathway plays important roles in a variety of cellular processes. Aberrant transduction of Notch signaling contributes to many diseases and cancers in humans. The Notch receptor intracellular domain, the activated form of Notch receptor, is extremely difficult to detect in normal cells. However, it can activate signaling at very low protein concentration to elicit its biological effects. In the present study, a cell based luciferase reporter gene assay was established in K562 cells to screen drugs which could modulate the endogenous CBF1‐dependent Notch signal pathway. Using this system, we found that the luciferase activity of CBF1‐dependent reporter gene was activated by baicalin and baicalein but suppressed by niclosamide in both dose‐ and time‐dependent manners. Treatment with these drugs modulated endogenous Notch signaling and affected mRNA expression levels of Notch1 receptor and Notch target genes in K562 cells. Additionally, erythroid differentiation of K562 cells was suppressed by baicalin and baicalein yet was promoted by niclosamide. Colony‐forming ability in soft agar was decreased after treatment with baicalin and baicalein, but was not affected in the presence of niclosamide. Thus, modulation of Notch signaling after treatment with any of these three drugs may affect tumorigenesis of K562 cells suggesting that these drugs may have therapeutic potential for those tumors associated with Notch signaling. Taken together, this system could be beneficial for screening of drugs with potential to treat Notch signal pathway‐associated diseases. J. Cell. Biochem. 106: 682–692, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

Dehydroascorbate reductase （DHAR） plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR （PbDHAR） from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcripUon-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coil following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity （19.84 i.mnol/min per mg） and high affinity （a Krn of 0.08 mM） towards the substrates dehydroascorbate （DHA）. Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55℃. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.     

Biophysical characterization of a new SCN5A mutation S1333Y in a SIDS infant linked to long QT syndrome

Various entities and genetic etiologies, including inherited long QT syndrome type 3 (LQT3), contribute to sudden infant death syndrome (SIDS). The goal of our research was to biophysically characterize a new SCN5A mutation (S1333Y) in a SIDS infant. S1333Y channels showed the gain of Na⁺ channel function characteristic of LQT3, including a persistent inward Na⁺ current and an enhanced window current that was generated by a −8 mV shift in activation and a +7 mV shift in inactivation. The correlation between the biophysical data and arrhythmia susceptibility suggested that the SIDS was secondary to the LQT3-associated S1333Y mutation.     

Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia

Mitochondria play important roles as the powerhouse of the cell. After cerebral ischemia, mitochondria overproduce reactive oxygen species (ROS), which have been thoroughly studied with the use of superoxide dismutase transgenic or knockout animals. ROS directly damage lipids, proteins, and nucleic acids in the cell. Moreover, ROS activate various molecular signaling pathways. Apoptosis-related signals return to mitochondria, then mitochondria induce cell death through the release of pro-apoptotic proteins such as cytochrome c or apoptosis-inducing factor. Although the mechanisms of cell death after cerebral ischemia remain unclear, mitochondria obviously play a role by activating signaling pathways through ROS production and by regulating mitochondria-dependent apoptosis pathways.     

Effect of blood pressure on vascular hemodynamics in acute tachycardia

Paroxysmal supraventricular tachycardia is accompanied by hypotension, which can affect vascular hemodynamics. Here, we hypothesized that a fall in blood flow as a result of hypotension has a larger effect on hemodynamics in medium-sized peripheral arteries compared with increased pulsatility in rapid pacing. To test this hypothesis, we experimentally and theoretically investigated hemodynamic changes in femoral, carotid, and subclavian arteries at heart rates of 95-170 beats/min after acute pacing. The arterial pressure, blood flow, and other hemodynamic parameters remained statistically unchanged for heart rates ≤ 135 beats/min. Systemic pressure and flow velocities, however, showed an abrupt decrease, resulting in larger alteration of hemodynamic parameters for heart rates ≥ 155 beats/min after pacing (initial period) and then recovered close to baseline after several minutes of pacing (recovery period). During the initial period, the pressure dropped from 88 mmHg (baseline) to 44 mmHg, and the flow velocity decreased to about one-third of baseline at heart rate of 170 beats/min. A hemodynamic analysis showed a velocity profile with a near-wall retrograde flow or a fully reversed flow during the initial period, which vanished at the recovery period. It was concluded that the initial fall of blood flow due to pressure drop led to transient flow reversal and negative wall shear stress because this phenomena was not observed at the recovery period. This study underscores the significant effects of hypotension on vascular hemodynamics, which may have relevance to physiology and chronic pathophysiology in paroxysmal supraventricular tachycardia.     

New insights into the effect of medium-chain-length lactones on yeast membranes. Importance of the culture medium

In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to γ-dodecalactone. After a 60-min exposure to 3 g L⁻¹, about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6 g L⁻¹ did not alter cultivability despite some transient loss of membrane integrity from 3 g L⁻¹. It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-β-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.     

HDM2 ERKs PCNA

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation.Almost 30 yr ago, proliferating cell nuclear antigen (PCNA) was first identified in dividing cells using sera derived from patients suffering from systemic lupus erythematosus (Takasaki et al., 1981). A few years later, the “mother” of all cancer markers had been associated with DNA synthesis (Madsen and Celis, 1985), but it wasn’t until 1988 that Bauer and Burgers (1988) and Prelich and Stillman (1988) discovered that the homotrimeric clamp served as a processivity factor for DNA polymerases. In 1992, Shivji et al. (1992) showed that PCNA was required for DNA repair, and 10 yr later, it was identified as a target of ubiquitin and SUMO (small ubiquitin-like modifier) conjugation after exposure to ultraviolet light (Hoege et al., 2002). For a protein that has been in the spotlight of modern biochemistry, it is quite remarkable that almost nothing is known about its normal cellular turnover.Insight into this process comes now from the study of an unlikely regulator. In this issue, Groehler and Lannigan (2010) demonstrate that the relatively poorly characterized ERK8 (extracellular signal-regulated kinase 8) takes center stage in the regulation of PCNA stability in primary mammary epithelial cells. The ERK family of kinases belongs to the mitogen-activated protein kinase superfamily and carries a Thr-Glu-Tyr (T-E-Y) activation motif that needs to be phosphorylated to enable kinase activity (Abe et al., 2002). Interestingly, ERK8 also needs to bind to chromatin to become active. The authors identified a highly conserved PXXXP motif in the C-terminal half of ERK8 that appeared to confer autoinhibition, an activity which is relieved upon chromatin binding. Relatively close by, in the middle of ERK8, resides a PCNA-interacting peptide (PIP) box required for the interaction with PCNA (Warbrick, 1998). Curiously, only the chromatin-bound fraction of ERK8 bound to the chromatin-bound fraction of PCNA. However, a functional PIP box was not required for ERK8 to associate with nuclear DNA in the cell. These results argue that ERK8 is not anchored to chromatin by PCNA but associates with it independently. Moreover, they strongly suggest that ERK8’s PIP box binds to PCNA only when the kinase is associated with chromatin. Importantly, overexpression of an ERK8 PIP box mutant resulted in destabilization of PCNA. The effect on PCNA stability seemed to be highly specific, as depletion of ERK8 caused codepletion of PCNA but did not lead to a decrease in steady-state levels of a variety of other cell cycle regulators.Why is the interaction with PCNA confined to chromatin? The reason is likely due to the fact that ERK8’s PIP box is buried in the middle of the protein. Most PCNA-interacting proteins carry their PIP box either at the N or C terminus (Vivona and Kelman, 2003). One other well-studied example for a protein with an internal PIP box is the essential replication factor MCM10 (minichromosome maintenance protein 10). MCM10 undergoes cell cycle–regulated modification, which probably induces a conformational switch that is necessary for the PIP box–mediated interaction with PCNA (Das-Bradoo et al., 2006). In the same vein, it is conceivable that chromatin association and the accompanying relief of autoinhibition of ERK8 cause the middle portion of the kinase to change its configuration, thereby assuming a functional PIP box domain that can be recognized by PCNA. In situations in which the rapid unloading of PCNA is required, regulation of ERK8 may be the most effective way to dispose of chromatin-bound PCNA, which is known to have an exceedingly low exchange rate (Sporbert et al., 2002). Despite the fact that interaction with ERK8 is necessary to stabilize chromatin-bound PCNA, it remains unclear whether PCNA is a direct target of ERK8-mediated phosphorylation.The next goal of Groehler and Lannigan (2010) was to dissect the mechanism underlying the ERK8-regulated degradation of PCNA. Based on the consideration that physical contact between the kinase and PCNA was an integral part of the protection, they hypothesized that ERK8 might compete with an E3 ubiquitin ligase that may target PCNA via its own PIP box. This turned out to be a smart guess because the only candidate to test was the E3 ligase HDM2, the human homologue of murine double minute 2 (Momand et al., 1992). In a set of well-controlled experiments, the authors not only demonstrate that HDM2 interacts directly with and degrades PCNA when ERK8 is absent, but they also exclude indirect effects by p53 and retinoblastoma (Rb) on this process. p53 is a direct target of HDM2 and is stabilized when their interaction is inhibited (Tao and Levine, 1999). Elevated levels of p53 trigger cell cycle arrest concomitant with hypophosphorylation of Rb, but none of these changes affect the stability of PCNA. It is not hard to imagine that the loss of chromatin-bound PCNA has severe consequences for the functionality of DNA replication and repair, resulting in chromosome breakage. The authors argued that a similar level of genome instability should be visible in ERK8-depleted cells. This was indeed the case as visualized by the accumulation of γ-H2AX foci and broken DNA (Rogakou et al., 1998). Importantly, Groehler and Lannigan (2010) observed similar effects in the ERK8 PIP box mutant, further lending credence to their model. It is worthwhile pointing out that the turnover of PCNA expands the spectrum of replication factors whose degradation is tightly linked to chromatin. CDT1, a member of the prereplication complex (Cook, 2009), is rapidly degraded in the face of DNA damage. Its degradation occurs exclusively on the chromatin-associated fraction of the protein pool and is dependent on CDT1 binding to PCNA (Arias and Walter, 2005; Hu and Xiong, 2006; Senga et al., 2006).An important question that this study raises is of course to what extent, if at all, is PCNA turnover deregulated in cancer cells? The commonly high levels of PCNA in transformed cells would be most compatible with a deregulation of ERK8 and/or HDM2 to provide a significant growth advantage. Indeed, the authors show in the last part of their study that in at least two transformed cell lines, PCNA is rendered inert to the presence of ERK8. They speculate that the underlying reason is a defect in HDM2, and although this is the most likely explanation, it still needs to be validated. It will be interesting to see how common the misregulation of PCNA turnover is in cancer tissues. At this point, it is intriguing to envision a dynamic scenario in which a two-step mechanism facilitates cell transformation (Fig. 1). Initially, deregulation of ERK8 may cause PCNA levels to decrease. This would contribute to genome instability and the accumulation of new mutations, including those affecting proper function of HDM2. In step two, deregulation of HDM2 may turn things around and result in an increase of PCNA, supporting rapid proliferation.Open in a separate windowFigure 1.Role of ERK8 in maintaining genome stability. (A) In normal cells, chromatin-bound ERK8 interacts with the chromatin fraction of PCNA, which resides at the replication fork (here just shown at the leading strand for simplicity). ERK8 binding inhibits the E3 ubiquitin ligase HDM2 from interacting with PCNA. (B) In cancer cells, inactivation of ERK8 enables HDM2 to interact with and ubiquitinate PCNA, targeting it for degradation. A decrease in PCNA levels causes an increase in DNA damage, resulting in the accumulation of new mutations. These new mutations may render HDM2 nonfunctional (rectangular form), which ultimately results in an increase of PCNA stability and facilitates cell proliferation. The homotrimeric PCNA structure (Protein Data Bank ID 2OD8) was generated using the Chimera software program (Pettersen et al., 2004).     

Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liver

To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2‐D PAGE with total, phospho‐ and glycoprotein staining and MALDI‐MS/MS and database searching were conducted. The results, based on fold‐change expression, revealed a down‐regulation of total protein expression by prenatal alcohol exposure in 7‐day‐old and 3‐month‐old rats. There was an up‐regulation of protein phosphorylation but a down‐regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo‐keto reductase, tumor rejection antigen gp96, fructose‐1,6‐bisphosphatase, glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, and γ‐actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S‐transferase, glucose regulated protein 58, α‐enolase, eukaryotic translation elongation factor 1 β‐2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3‐hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications.     

Damage-specific modification of PCNA

Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not necessarily have to occur during S phase, but could be postponed to late in S phase after the bulk of DNA synthesis had been completed. This view was primarily based on the arrest phenotype of temperature-sensitive DNA ligase I mutants in yeast, which accumulated with an almost fully duplicated set of chromosomes. However, many temperature-sensitive alleles can be leaky and the re-evaluation of DNA ligase I-deficient cells has offered new and unexpected insights into how cells keep track of lagging strand synthesis. It turns out that if Okazaki fragment joining goes awry, cells have their own alarm system in the form of ubiquitin that is conjugated to the replication clamp PCNA. Although this modification results in mono- and poly-ubiquitination of PCNA, it is genetically distinct from the known post-replicative repair mark at lysine 164. In this Extra View, we discuss the possibility that eukaryotic cells utilize different enzymatic pathways and ubiquitin attachment sites on PCNA to alert the replication machinery to the accumulation of single-stranded gaps or nicks behind the fork.Key words: DNA ligase I, DNA replication, Okazaki fragment processing, PCNA, ubiquitin, SUMO