Phosphatase Binding Research Articles

Differential effect of apoptosis‐associated tyrosine kinase on K‐Cl cotransporter activity in C6 glioblastoma cells (694.2)

Expression of the potassium‐chloride (K‐Cl) cotransporter is linked to the growth and spread of gynecological cancers. Immunofluorescent localization of three K‐Cl cotransporter isoforms (KCC1, KCC3, and KCC4) to migrating extensions of rat glioblastoma (C6) cells suggests their potential role in the enhanced motility of these tumors. RT‐PCR amplification of mRNA from C6 cells validated the expression of kinases and phosphatases known to control cation‐chloride cotransporter activity. Xenopus laevis oocytes co‐injected with KCC3 and apoptosis‐associated tyrosine kinase (AATYK1) cRNA showed isosmotic K+ influx similar to hyposmotically‐activated K+ influx, whereas no change in isosmotic K+ influx was observed with co‐injection of KCC1 and AATYK1 cRNA. This differential effect of AATYK1 on two KCC isoforms prompted testing of the potential of AATYK1 as a novel target for affecting glioblastoma motility. Genistein, a general tyrosine kinase inhibitor, failed to reduce the hyposmotically‐stimulated K+ influx of C6 cells or inhibit their motility in a scratch wound assay indicating that the catalytic activity of the tyrosine kinase is not involved. Molecular identification of both serine/threonine kinase and phosphatase binding sites on AATYK1 suggests that it may scaffold other K‐Cl cotransporter regulatory proteins and thus represent a novel target for anti‐cancer therapeutics.Grant Funding Source: Supported by a Special Projects Grant from the University of Saskatchewan College of Medicine

A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts

Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi.

Exenatide can inhibit calcification of human VSMCs through the NF-kappaB/RANKL signaling pathway

Arterial calcification is an important pathological change of diabetic vascular complication. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important cytopathologic role in arterial calcification. The glucagon-like peptide-1 receptor agonists (GLP-1RA), a novel type of antidiabetic drugs, exert cardioprotective effects through the GLP-1 receptor (GLP-1R). However, the question of whether or not GLP-1RA regulates osteoblastic differentiation and calcification of VSMCs has not been answered, and the associated molecular mechanisms have not been examined. Calcifying VSMCs (CVSMCs) were isolated from cultured human arterial smooth muscle cells through limiting dilution and cloning. The extent of matrix mineralization was measured by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot. Gene expression of receptor activator of nuclear factor-κB ligand (RANKL) was silenced by small interference RNA (siRNA). Exenatide, an agonist of GLP-1 receptor, attenuated β-glycerol phosphate (β-GP) induced osteoblastic differentiation and calcification of human CVSMCs in a dose- and time-dependent manner. RANKL siRNA also inhibited osteoblastic differentiation and calcification. Exenatide decreased the expression of RANKL in a dose-dependent manner. 1,25 vitD3 (an activator of RANKL) upregulated, whereas BAY11-7082 (an inhibitor of NF-κB) downregulated RANKL, alkaline phosphatase (ALP), osteocalcin (OC), and core binding factor α1 (Runx2) protein levels and reduced mineralization in human CVSMCs. Exenatide decreased p-NF-κB and increased p-AMPKα levels in human CVSMCs 48 h after treatment. Significant decrease in p-NF-κB (p-Ser276, p-Ser536) level was observed in cells treated with exenatide or exenatide++κBAY11-7082. GLP-1RA exenatide can inhibit human VSMCs calcification through NF-κB/RANKL signaling.

MEK1 and MEK2 differentially control the duration and amplitude of the ERK cascade response

The Raf/MEK/ERK cascade is one of the most studied and important signal transduction pathways. However, existing models largely ignore the existence of isoforms of the constituent kinases and their interactions. Here, we propose a model of the ERK cascade that includes heretofore neglected differences between isoforms of MEK. In particular, MEK1 is subject to a negative feedback from activated ERK, which is further conferred to MEK2 via hetero-dimerization. Specifically, ERK phosphorylates MEK1 at the residue Thr292, hypothetically creating an additional phosphatase binding site, accelerating MEK1 and MEK2 dephosphorylation. We incorporated these recently discovered interactions into a mathematical model of the ERK cascade that reproduces the experimental results of Catalanotti et al (2009 Nature Struct. Mol. Biol. 16 294–303) and Kamioka et al (2010 J. Biol. Chem. 285 33540–8). Furthermore, the model allows for predictions regarding the differences in the catalytic activity and function of the MEK isoforms. We propose that the MEK1/MEK2 ratio regulates the duration of the response, which increases with the level of MEK2 and decreases with the level of MEK1. In turn, the amplitude of the response is controlled by the total amount of the two isoforms. We confirm the proposed model structure performing a random parameter sampling, which led us to the conclusion that the sampled parameters, selected to properly reproduce wild-type (WT) cell behavior, to allow for qualitative reproduction of differences in behavior WT cells and cell mutants studied experimentally.

Transcription Factor Sp3 Represses Expression of p21CIP1 via Inhibition of Productive Elongation by RNA Polymerase II

Like that of many protein-coding genes, expression of the p21(CIP1) cell cycle inhibitor is regulated at the level of transcription elongation. While many transcriptional activators have been shown to stimulate elongation, the mechanisms by which promoter-specific repressors regulate pausing and elongation by RNA polymerase II (RNA PolII) are not well described. Here we report that the transcription factor Sp3 inhibits basal p21(CIP1) gene expression by promoter-bound RNA PolII. Knockdown of Sp3 led to increased p21(CIP1) mRNA levels and reduced occupancy of the negative elongation factor (NELF) at the p21(CIP1) promoter, although the level of binding of the positive transcription elongation factor b (P-TEFb) kinase was not increased. Sp3 depletion correlated with increased H3K36me3 and H2Bub1, two histone modifications associated with transcription elongation. Further, Sp3 was shown to promote the binding of protein phosphatase 1 (PP1) to the p21(CIP1) promoter, leading to reduced H3S10 phosphorylation, a finding consistent with Sp3-dependent regulation of the local balance between kinase and phosphatase activities. Analysis of other targets of Sp3-mediated repression suggests that, in addition to previously described SUMO modification-dependent chromatin-silencing mechanisms, inhibition of the transition of paused RNA PolII to productive elongation, described here for p21(CIP1), is a general mechanism by which transcription factor Sp3 fine-tunes gene expression.

Pyruvate restores β-adrenergic sensitivity of L-type Ca2+channels in failing rat heart: role of protein phosphatase

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

Essential Roles of the Tap42-Regulated Protein Phosphatase 2A (PP2A) Family in Wing Imaginal Disc Development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.

Effects of Glucocorticoid Receptor Small Interfering RNA Delivered Using Poly Lactic-Co-Glycolic Acid Microparticles on Proliferation and Differentiation Capabilities of Human Mesenchymal Stromal Cells

Bone marrow-derived mesenchymal stem cells (MSC) are a potential attractive source of cells for stem cell-based tissue regeneration, but the small number and reduced capabilities of MSC proliferation and differentiation due to in vitro replicative senescence and donor-associated pathophysiological factors, including age and estrogen depletion, severely restrict their potential usefulness in clinical applications. Glucocorticoids (GC) are well-known steroid hormones that regulate MSC proliferation and differentiation, but the defined effects and underlying mechanisms of endogenous glucocorticoids on MSC characteristics are not understood. This study investigated the effects of the blockage of endogenous GC using glucocorticoid receptor (GR) small interfering RNA (siRNA) delivered using biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles on proliferation and differentiation capabilities of human MSC in vitro. The results show that we can prepare PLGA microparticles as a delivery system for GR siRNA and maintain release of siRNA up to 40 days in vitro. Transfection of GR siRNA significantly downregulates GR and upregulates the expression of fibroblast growth factor-2 and Sox-11 of human MSC. MSC that have proliferated with endogenous GC blocked in vitro have greater proliferation rates and exhibit upregulated expression of osteogenic markers (alkaline phosphatase and core binding factor alpha 1) under differentiation stimulation after 1 week. Under adipogenic differentiation, MSC proliferated in vitro with siRNA transfection, resulting in significantly lower adipogenic markers (peroxisome proliferator-activated receptor and lipoprotein lipase) than controls. In conclusion, PLGA particles can serve as a tool for delivery of GR siRNA to effectively block the effects of endogenous GC on MSC, which has the potential to improve the capabilities of human MSC for clinical application by preventing replicative senescence.

Drosophila spoonbill encodes a dual-specificity A-kinase anchor protein essential for oogenesis

spoonbill is a Drosophila female-sterile mutation, which interferes with normal egg patterning during oogenesis. Previous analyzes linked the mutation to a number of seemingly unrelated pathways, including GRK/EGFR and DPP, two major pathways essential for Drosophila and vertebrate development. Further work suggested that spoonbill may also function in actin polymerization and border-cell migration. Here we describe the molecular cloning of the spoonbill gene and characterize new mutant alleles, further demonstrating that spoonbill’s function is essential during oogenesis. We found spoonbill to be allelic to CG3249 (also known as yu), which encodes the only known dual-specificity A-kinase anchor protein in Drosophila. Our data indicate that similar to mammalian AKAPs, Spoonbill protein contains a number of potential kinase and phosphatase binding motifs, and is targeted, in the ovary, to mitochondria and Golgi. Finally, we address some of spoonbill’s mutant phenotypes from the perspective of the published data on the AKAP protein family.

KNL1/Spc105 Recruits PP1 to Silence the Spindle Assembly Checkpoint

The spindle assembly checkpoint (SAC) delays anaphase onset until kinetochores accomplish bioriented microtubule attachments [1]. Although several centromeric and kinetochore kinases, including Aurora B, regulate kinetochore-microtubule attachment and/or SAC activation [2-4], the molecular mechanism that translates bioriented attachment into SAC silencing remains unclear [5]. Employing a method to rapidly induce exact gene replacement in budding yeast [6], we show here that the binding of protein phosphatase 1(PP1/Glc7) to the evolutionarily conserved RVSF motif of the kinetochore protein Spc105 (KNL1/Blinkin/CASC5) is essential for viability by silencing the SAC, while it plays an auxiliary nonessential role for physical chromosome segregation. Although Aurora B may inhibit this binding, persistent PP1-Spc105 interaction does not affect chromosome segregation and is insufficient to silence the SAC in the absence of microtubules, indicating that dynamic regulation of thisinteraction is dispensable. However, the amount of PP1 targeted to kinetochores must be finely tuned, because recruitment of either no or one extra copy of PP1 to Spc105 is detrimental, illustrating the vital impact of targeting an exiguous fraction of PP1 to the kinetochore. We propose that the PP1-Spc105 interaction enables local regulation of dynamic phosphorylation and dephosphorylation at the kinetochore to couple microtubule attachment and SAC silencing.

Survivin (BIRC5) cell cycle computational network in human no‐tumor hepatitis/cirrhosis and hepatocellular carcinoma transformation

Survivin (BIRC5) relationship with tumor is presented in several papers. However, how the molecular network and interpretation concerning BIRC5 cell cycle between no-tumor hepatitis/cirrhosis and hepatocellular carcinoma (HCC) remains to be elucidated. Here, we constructed and analyzed significant higher expression gene BIRC5 activated and inhibited cell cycle network from HCC versus no-tumor hepatitis/cirrhosis patients (viral infection HCV or HBV) in GEO Dataset by combination of gene regulatory network inference method based on linear programming and decomposition procedure with the CapitalBio MAS 3.0 software based on the integration of public databases including Gene Ontology, KEGG, BioCarta, GenMapp, Intact, UniGene, OMIM, etc. Compared the same and different activated and inhibited BIRC5 network with GO analysis between no-tumor hepatitis/cirrhosis and HCC, our result showed BIRC5 cell cycle network weaker transcription factor activity in both no-tumor hepatitis/cirrhosis and HCC (1); stronger nucleus protein binding but weaker cytoplasm protein binding in no-tumor hepatitis/cirrhosis (2); stronger cytoplasm protein phosphatase binding but weaker ubiquitin-protein ligase activity in HCC (3). Therefore, we inferred BIRC5 cell cycle module less transcription from RNA polymerase II promoter in both no-tumor hepatitis/cirrhosis and HCC (4). We deduced BIRC5 cell cycle module different from more mitosis but less complex-dependent proteasomal ubiquitin-dependent protein catabolism as a result increasing cell division and cell numbers in no-tumor hepatitis/cirrhosis to more protein amino acid autophosphorylation but less negative regulation of ubiquitin ligase activity during mitotic cell cycle as a result increasing growth and cell volume in HCC (5).

Tumor Promoters - Microcystin-LR, Nodularin and TNF-α and Human Cancer Development

Microcystin-LR and nodularin, along with okadaic acid, are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The mechanisms of action of microcystin-LR and nodularin in the liver and that of okadaic acid, a potent tumor promoter on mouse skin, have attracted the attention of the scientists. This paper reviews several topics: new inhibitors of PP1 and PP2A with new chemical structures, structure-function relationships for both receptor binding and inhibition of protein phosphatases, the crystal structure of PP1 or PP2A-toxin complex, induction of gene expression and apoptosis. These subjects were studied by using in vitro and in vivo experimental systems. Two-stage carcinogenesis experiments with microcystin-LR and nodularin for the first time demonstrated that microcystin-LR is a new tumor promoter in rat liver initiated with diethylnitrosamine (DEN), and that nodularin is a potent tumor promoter associated with weak initiating activity in rat liver initiated with DEN. A working group of WHO (IARC) concluded that microcystin-LR is "possibly carcinogenic to humans" and that nodularin is "not classifiable as to carcinogenicity". Our studies revealed that chemical tumor promoters are inducers of TNF-α in the cells of target tissues and that TNF-α is an endogenous tumor promoter. This advance in carcinogenesis made it possible to look for the link between chemical tumor promoters and endogenous tumor promoters, such as TNF-α and IL-1. The carcinogenic features of TNF-α are described in this review, and the TNF-α inducing protein (Tipα) of Helicobacter pylori genome is presented as an example of a tumor promoter of human stomach cancer development.

Eicosapentaenoic acid reduces warfarin-induced arterial calcification in rats

BackgroundEicosapentaenoic acid (EPA), a major n-3 polyunsaturated fatty acid, is reported to have various protective effects for cardiovascular disease. However, few studies have focused on the influence of EPA on vascular calcification. Methods and resultsArterial medial calcification (AMC) was induced by administering warfarin (3mg/g food) and vitamin K1 (1.5mg/g food) for 2 weeks in Sprague–Dawley rats (control group), and EPA (1g/kg/day) was administered for 2 weeks simultaneously with warfarin and vitamin K1 (EPA group) or after initiation of AMC (late EPA group). EPA showed a marked reduction of medial calcification in the EPA group, and showed a similar effect in the late EPA group. Immunohistochemical and RT-PCR analyses showed that EPA lowered the expression of osteogenetic markers, such as osteopontin, alkaline phosphatase and core binding factor-α1 in the aorta. Significant migration of macrophages with expression of matrix-metalloproteinase (MMP)-2 or MMP-9 was observed in the aortic adventitia around calcification. EPA also reduced macrophage infiltration, MMP-9 expression as well as gene expression of monocyte chemotactic protein (MCP)-1. ConclusionsThese observations indicate that EPA attenuates arterial medial calcification through an effect associated with the suppression of MMP-9 activity and inhibition of macrophage infiltration as well as osteogenic protein expression in warfarin-induced rat models.

Coupling between the voltage-sensing and phosphatase domains of Ci-VSP

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

Control of Cellular GADD34 Levels by the 26S Proteasome

GADD34, the product of a growth arrest and DNA damage-inducible gene, is expressed at low levels in unstressed cells. In response to stress, the cellular content of GADD34 protein increases and, on termination of stress, rapidly declines. We investigated the mechanisms that control GADD34 levels in human cells. GADD34 proteins containing either an internal FLAG or a C-terminal green fluorescent protein epitope were degraded at rates similar to endogenous GADD34. However, the addition of epitopes at the N terminus or deletion of N-terminal sequences stabilized GADD34. N-terminal peptides of GADD34, either alone or fused to heterologous proteins, exhibited rapid degradation similar to wild-type GADD34, thereby identifying an N-terminal degron. Deletion of internal PEST repeats had no impact on GADD34 stability but modulated the binding and activity of protein phosphatase 1. Proteasomal but not lysosomal inhibitors enhanced GADD34 stability and eukaryotic initiation factor 2alpha (eIF-2alpha) dephosphorylation, a finding consistent with GADD34's role in assembling an eIF-2alpha phosphatase. GADD34 was polyubiquitinated, and this modification enhanced its turnover in cells. A stabilized form of GADD34 promoted the accumulation and aggregation of the mutant cystic fibrosis transmembrane conductance regulator (CFTRDeltaF508), highlighting the physiological importance of GADD34 turnover in protein processing in the endoplasmic reticulum and the potential impact of prolonged GADD34 expression in human disease.

RNA interference of the BMPR‐IB gene blocks BMP‐2‐induced osteogenic gene expression in human bone cells

We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo.

Clinical Utility of Malnutrition-Inflammation Score in Maintenance Hemodialysis Patients: Focus on Identifying the Best Cut-Off Point

Background: Malnutrition-inflammation score (MIS) is a comprehensive and quantitative system to assess malnutrition-inflammation complex syndrome, and a strong correlation between MIS and morbidity/mortality in maintenance hemodialysis (MHD) patients had been demonstrated. However, there is no cut-off value of MIS to categorize patients into high risk or low risk patients. Methods: A total of 257 chronic stable and ambulatory adult MHD patients from Far Eastern Memorial Hospital were enrolled for the study. The MIS of each patient was recorded at the initiation of study and the study population was followed up as a 12-month prospective cohort to evaluate mortality as the primary outcome. Results: Twelve patients died in the 12-month observational period. Both multiple logistic regression analyses and Cox proportional hazards model denoted MIS, alkaline phosphatase, transferrin saturation, ferritin, and total iron binding capacity as significant predictors of 1-year mortality. The conditional effect plot of MIS on 1-year mortality revealed that when fixing the alkaline phosphatase, transferrin saturation, ferritin, and total iron binding capacity at a mean value, the probability of death for an MHD patient whose MIS was 3, 4, and 5 is 10, 40, and 80%, respectively. Conclusions: Our study shows that MHD patients with MIS score of more than 4–5 had a significant risk of 1-year mortality. Additional risk factors associated with short-term mortality besides malnutrition-inflammation complex syndrome were anemia and renal osteodystrophy. This study proves that MIS is a useful tool to risk-stratify Asian MHD patients and to identify those at risk of short-term death. Nutritional interventions that can improve the MIS may also improve survival, but this hypothesis needs to be verified in interventional studies.

2-Methoxyestradiol inhibits hepatocellular carcinoma cell growth by inhibiting Cdc25 and inducing cell cycle arrest and apoptosis

2-Methoxyestradiol (2-ME) is a physiological metabolite of estrogen, which can inhibit growth of many types of tumor cells, including hepatocellular carcinoma, both in vitro and in vivo. The exact mechanisms of its action are still unclear. We have studied the mechanisms of growth inhibition of several of human and rat hepatoma and normal liver cells by 2-ME. Human (Hep3B, HepG2, PLC/PRF5) and rat (McA-RH7777, JM-1) hepatoma and normal rat (CRL-1439) and human (CRL-11233) liver cell lines were cultured in vitro, in presence of 2-ME, and its IC50s were determined. Cell cycle arrest, Cdc25 phosphatase inhibition and apoptosis induction were studied. Finally, the effect of 2-ME on the growth of JM-1 rat hepatoma cells in rat liver was determined in vivo. The IC50 range for growth inhibition of hepatoma cells was found to be between 0.5 and 3 microM. In contrast, normal rat hepatocytes and liver cell lines were resistant to 2-ME up to 20 microM. JM-1 cells were arrested in the G2/M phase of the cell cycle. Cdc25A and Cdc25B, cell cycle controlling phosphatases, activities were inhibited in vitro and 2-ME was found to likely bind to their catalytic site cysteines. As a consequence, their cellular substrates Cdk1 and Cdk2 were tyrosine phosphorylated. Caspase-3 was cleaved suggesting apoptotic cell death. Moreover, growth of JM-1 tumors, which were transplanted into rat liver, was also inhibited by treatment with 2-ME in vivo. 2-Methoxyestradiol is a selective, potent and relatively non-toxic hepatoma growth inhibitor both in vitro and in vivo. Cell cycle arrest of hepatoma cells was likely mediated by binding and inactivation of the Cdc25 phosphatases and induction of apoptosis.

Electrochemical detection of point mutation based on surface ligation reaction and biometallization

A highly sensitive electrochemical method for point mutation detection based on surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface-immobilized allele-specific probe, complementary to the mutant target, undergoes allele-specific ligation with the 5′-phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90°C, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, a non-reductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AA-P), can be converted into ascorbic acid (AA) at the electrode surface. Silver ions in solution are then reduced by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep voltammetry (LSV) is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K-ras oncogene target with a detection limit of 80fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.

Role of a Founder c.201_202delCT Mutation and New Phenotypic Features of Congenital Lipoid Adrenal Hyperplasia in Palestinians

Congenital lipoid adrenal hyperplasia (CLAH), caused by mutations in steroidogenic acute regulatory protein (StAR), is most frequent in Japanese and Palestinians. We report eight Palestinians from four unrelated families with CLAH. The objective of the study was to identify the mutation(s) in StAR, correlate genotype with phenotype, and determine whether the common mutation represents a founder mutation. Clinical, histopathological, and molecular genetic characterization was performed in these eight patients. All affected individuals (three XY, five XX) presented neonatally with undetectable adrenocortical hormones and are responding to replacement therapy. Only two sisters had neurodevelopmental deficits. Histopathological findings of excised XY gonads included accumulation of fat in Leydig cells. Significantly, already at 1 yr of age, positive placental alkaline phosphatase and octamer binding transcription factor staining indicated neoplastic potential. Sequence analysis of StAR revealed homozygosity for c.201_202delCT mutation in all eight cases, causing premature termination of the StAR protein. This mutation was confirmed to be a founder mutation using both an intragenic microsatellite and several single nucleotide polymorphism markers. Screening of 100 normal Jerusalem Palestinians detected no carriers of this mutation. CLAH is rare in the general Palestinian population. In most Palestinian cases, a founder c.201_202delCT mutation in StAR is the cause. The observed early neonatal presentation may reflect the major StAR protein truncation caused by this mutation. A crucial role for StAR in the central nervous system was not supported with normal neurological examinations in six of eight cases. Finally, we advocate early gonadectomy in XY CLAH cases, given the early onset of neoplastic changes observed histologically.