BJ Disease Immediate Publications

Readthrough of long-QT syndrome type 1 nonsense mutations rescues function but alters the biophysical properties of the channel

S C Harmer, J S Mohal, D Kemp, A Tinker — 2012-02-06T15:18:15Z

The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause long-QT syndrome type 1 (LQT1) and result in a complete loss of I_Ks channel function. In this study we attempted to rescue the function of these mutants, in human embryonic kidney-293 cells, by promoting readthrough of their premature termination codons (PTCs) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418, acted to promote full-length channel protein expression from R518X at 100 µM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV, for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced, from R518X, while similar were not identical to wild-type as the voltage dependence of activation was significantly (P<0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.

Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex

L Troeberg, B Mulloy, P Ghosh, M Lee, G Murphy, H Nagase — 2012-02-03T11:43:36Z

The semi-synthetic sulfated polysaccharide pentosan polysulfate (PPS) increases affinity between the aggrecan-degrading adamalysins with thrombospondin motifs (ADAMTSs) and their endogenous inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3. Here we demonstrate that PPS mediates the formation of a high affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular matrix binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Spacer domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of 8 saccharide units, indicating the involvement of extended or multiple protein interaction sites. The formation of a high affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

p53Ser15 Phosphorylation disrupts p53-RPA70 complex and induces RPA70-mediated DNA repair in hypoxia

E Madan, R Gogna, U Pati — 2012-01-30T13:47:24Z

Cellular stressors are known to inhibit p53-RPA70 complex and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for inhibition of apoptosis in hypoxic tumors. We have shown that, in cancer cells, hypoxia disrupts p53-RPA70 complex thereby enhancing RPA70-mediated NER/NHEJ-repair. In normal cells, RPA70 binds to p53 N-terminus (NTD) whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in NTD confirm that p53^Ser15phosphorylation induces dissociation of p53-RPA70 complex in hypoxia. DNA-PK is shown to induce p53^Ser15phosphorylation thus enhancing RPA70-mediated NER/NHEJ-repair. Further, RPA70 gene silencing induces significant increase in the cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53^Ser15 phosphorylation dissociates p53-RPA70 complex in enhancing NER/NHEJ-repair that causes resistance to apoptosis, in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics based upon regulation of RPA70-mediated NER/NHEJ-repair.

First identification of small molecule inhibitors of Pontin by combining virtual screening and enzymatic assay

2012-01-24T14:13:58Z

The human protein Pontin, which belongs to the AAA+ family, is overexpressed in several cancers and its silencing in vitro leads to tumor cell growth arrest and apoptosis, making it a good target for cancer therapy. In particular, high levels of expression were found in hepatic tumors for which the therapeutic arsenal is rather limited. The 3D structure of Pontin had previously been resolved, revealing an hexameric assembly with one ADP molecule co-crystallized in each subunit. Using Vina, Drugscore and Xscore, structure-based virtual screening of 2,200 commercial molecules was conducted into the ATP binding site formed by a dimer of Pontin in order to prioritize the best candidates. Complementary to the in silico screening, a versatile and sensitive colorimetric assay was set up to measure the disruption of the ATPase activity of Pontin. This assay allowed the determination of inhibition curves for more than twenty top scoring compounds, resulting in the identification of four ligands presenting an inhibition constant in the micromolar concentration range. Three of them inhibited tumor cell proliferation. The association of virtual screening and experimental assay thus proved successful for the discovery of the first small molecule inhibitors of Pontin.

The nonsteroidal anti-inflammatory drug indomethacin activates the eIF2alpha kinase PKR, causing a translational block in human colorectal cancer cells

C Brunelli, C Amici, M Angelini, C Fracassi, G Belardo, M Santoro — 2012-01-23T14:57:01Z

The non-steroidal anti-inflammatory drug (NSAID) indomethacin, a cyclooxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against colorectal cancer (CRC) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. Herein we show that indomethacin selectively activates the double-stranded RNA (dsRNA)-dependent protein kinase PKR in a cyclooxygenase-independent manner, causing rapid phosphorylation of the alpha-subunit of eukaryotic translation initiation-factor 2 (eIF2alpha) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of eIF2alpha-kinases PERK, GCN2 and HRI, and induced eIF2alpha phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in-vitro, an effect reversed by PKR-inhibitor 2-aminopurine, suggests a direct effect of the drug in the kinase activation. The results identify PKR as a novel target of indomethacin, opening new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.

Transforming Growth Factor Beta-1 represses proximal tubular cell microRNA-192 expression via decreased Hepatocyte Nuclear Factor DNA binding

R H. Jenkins, J Martin, A O. Phillips, T Bowen, D J. Fraser — 2012-01-23T12:05:33Z

MicroRNA-192 (miR-192) plays key roles in renal pathological and physiological responses, by repressing targets including Zeb1, Zeb2, and Wnk1. Here, we have studied regulation of miR-192 expression. We found that Transforming Growth Factor Beta-1 (TGF-b1) down-regulates miR-192 and miR-194, co-transcribed in the shared precursor pri-miR-192/194. Luciferase reporter analysis showed constitutive promoter activity within nucleotides +21 to -223. We identified Hepatocyte Nuclear Factor and p53 binding sites within this region that were required for constitutive promoter activity, which was decreased by TGF-b1 via an Alk5-dependent mechanism. TGF-b1-treatment decreased HNF binding to the miR-194-2/192 promoter, while knockdown of HNF-1 inhibited mature miR-192 and -194 expression. miR-192, -194 and HNF expression were restricted to a defined subset of human tissues including kidney, small intestine, colon, and liver. Our data identify coordinated regulation of miR-192 and -194, with binding of HNF and p53 transcription factors necessary for activation of transcription, and TGF-b1 mediated repression via decreased HNF binding to its cognate promoter element.

Proteoglycans of uterine fibroids and keloid scars: similarity in their proteoglycan composition

D A. Carrino, S Mesiano, N M. Barker, W W. Hurd, A I. Caplan — 2012-01-19T11:22:29Z

Fibrosis is the formation of excess and abnormal fibrous connective tissue as a result of either a reparative or reactive process. A defining feature of connective tissue is its extracellular matrix, which provides structural support and also influences cellular activity. Two common human conditions that result from fibrosis are uterine fibroids (leiomyomas) and keloid scars. Because these conditions share a number of similarities and because their growth is due primarily to excessive extracellular matrix deposition, we compared the proteoglycans of uterine fibroids and keloid scars to corresponding normal tissues. Our analysis indicates that uterine fibroids and keloid scars contain higher amounts of proteoglycans relative to normal myometrium and normal adult skin, respectively. Proteoglycan composition is also different in the fibrotic tissues. Compared to unaffected tissues, uterine fibroids and keloid scars contain higher relative amounts of versican and lower relative amounts of decorin. There is also evidence for a higher level of versican catabolism in the fibrotic tissues compared to unaffected tissues. These qualitative and quantitative proteoglycan differences may play a role in the expansion of these fibrotic conditions and in their excessive matrix deposition and matrix disorganization due to effects on cell proliferation, TGF-β signaling, and/or collagen fibril formation.

The antineurodegenerative agent clioquinol regulates the transcription factor FOXO1a

A R Cameron, K Wallace, L Logie, A R Prescott, T G Unterman, J Harthill, G Rena — 2012-01-16T14:43:21Z

Many diseases of ageing including Alzheimer’s Disease (AD) and type 2 diabetes (T2D) are strongly associated with common risk factors, such as hypertension, hyperglycaemia and hyperinsulinaemia, suggesting that there may be shared ageing mechanisms underlying these diseases, with scope to identify common cellular targets for therapy. Here we have studied insulin-like signalling properties of an experimental AD 8-hydroxyquinoline drug known as clioquinol. The insulin/IGF-1 signal transduction (IIS) kinase Akt/PKB inhibits the transcription factor FOXO1a by phosphorylating it on residues that trigger its exit from the nucleus and in 293 cells we found that clioquinol treatment induces similar effects. A key transcriptional response to IIS is inhibition of hepatic gluconeogenic gene expression and in rat liver cells, clioquinol represses expression of the key gluconeogenic regulatory enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). The effects on FOXO1a and gluconeogenic gene expression require the presence of zinc ions, reminiscent of much earlier literature examining diabetogenic properties of 8-hydroxyquinolines. Comparative investigation of the signalling properties of a panel of these compounds demonstrates that CQ alone exhibits FOXO1a regulation without diabetogenicity. Our results suggest that zinc-dependent regulation of FOXOs and gluconeogenesis may contribute to the therapeutic properties of this drug. Further investigation of this signalling response might illuminate novel pharmacological strategies for the treatment of age-related diseases.

The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference

2012-01-16T12:05:38Z

The malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal-5-phosphate (PLP), the active form of vitamin B6. We show here that the de novo synthesized PLP is used by the parasite to detoxify singlet molecular oxygen (¹O₂), a highly destructive reactive oxygen species arising from hemoglobin digestion. The formation of singlet oxygen and the response of the parasite were monitored by life cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite, respectively. Pull-down experiments of transgenic parasites overexpressing the vitamin B6 biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 microM in wild-type parasites to 36.6 microM in the Pdx1/Pdx2-overexpressing cells and thus to a higher tolerance towards ¹O₂. In contrast, by applying cellular protein interference using inactive Pdx1 and Pdx2 mutants P. falciparum became susceptible to singlet oxygen. Our results clearly demonstrate the crucial role of vitamin B6 biosynthesis in the detoxification of singlet oxygen in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes this second important task of the vitamin B6 de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

Transcriptional regulation mechanisms of Hypoxia-induced neuroglobin gene expression

N liu, Z Yu, S Xiang, S Zhao, A Tjärnlund Wolf, C Xing, J Zhang, X Wang — 2012-01-13T11:45:00Z

Neuroglobin (Ngb) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulation mechanisms of Ngb expression, especially under hypoxia conditions. In this study, we located the core proximal promoter of mouse Ngb gene to a 554 bp segment, which harbors putative conserved NFκB, Egr1 binding sites. Over-expression and knock-down of transcription factors p65, p50, Egr1 or Sp1 increased and decreased Ngb expression, respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA and ChIP assays demonstrated that NFκB family members (p65, p50, cRel), Egr1, and Sp1 bound in vitro and in vivo to the proximal promoter region of Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NFκB (p65) and Sp1 were also responsible for hypoxia-induced upregulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of mouse Ngb gene, our results suggested that HIF1α is also involved in hypoxia-induced Ngb upregulation. In conclusion, we identified that NFκB, Egr1, and Sp1 played important roles in regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NFκB, Sp1 and HIF1α contributed to the upregulation of mouse Ngb gene expression under hypoxic conditions.

Protein kinase D2 has a restricted but critical role in T cell antigen receptor signaling in mature T cells

2012-01-10T16:33:45Z

Protein Kinase D2 (PKD2) is a serine/threonine kinase activated by diacylglycerol in response to engagement of antigen receptors in lymphocytes. To explore PKD2 regulation and function in T cell antigen receptor (TCR) signal transduction we expressed TCR complexes with fixed affinity for self antigens in T cells of PKD2 null mice or mice deficient in PKD2 catalytic activity. We also developed a single cell assay to quantify PKD2 activation as T cells respond to developmental stimuli or engagement of a/b TCR complexes in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T cell development is thus critical to regulate thymic homeostasis. PKD2 null T cells that exit the thymus have a normal transcriptome but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective but critical function for PKD2 in signaling by a/b mature TCR complexes in peripheral T cells.

Human protein derived peptides for intracellular delivery of biomolecules

A K Haas, D Maisel, J Adelmann, C von Schwerin, I Kahnt, U Brinkmann — 2011-12-13T11:54:08Z

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve cell penetrating peptides (CPPs) or protein transduction domains. Most of these require rather high concentrations to elicit cell penetrating functionality, are non-human, pathogen-derived or synthetic entities and may therefore not be tolerated or even immunogenic. We identified novel human protein derived CPPs by a combination of in-silico and experimental analyses: polycationic CPP candidates were identified in an in-silico library of all 30mer peptides of the human proteome. 60 of these peptides derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA transfection assays revealed that 20 of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human Neurturin (NRTN) that contains an alpha helix performed best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.

Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein-D: Implications for lipid antioxidant activity and Alzheimer{'}s disease

S Bhatia, B Knoch, J Wong, W Scott Kim, P L. Else, A J Oakley, B Garner — 2011-12-12T15:05:50Z

Apolipoprotein-D (apoD) is upregulated in Alzheimer’s disease (AD) and upon oxidative stress. ApoD inhibits brain lipid peroxidation in vivo but the mechanism is unknown. Specific Met residues may inhibit lipid peroxidation by reducing radical-propagating lipid hydroperoxides to non-reactive hydroxides via a reaction that generates methionine sulfoxide (MetSO). Since apoD has three conserved Met residues (M49, M93, M157), we generated recombinant proteins with either one or all Met residues replaced by Ala and assessed their capacity to reduce hydroperoxyeicosatetraenoic acids (HpETEs) to their hydroxyeicosatetraenoic acid (HETE) derivatives. ApoD, apoD(M49-A) and apoD(M157-A) all catalysed the reduction of HpETEs to their corresponding HETEs. Amino acid analysis of HpETE-treated apoD revealed a loss of one third of the Met residues accompanied by the formation of MetSO. Additional studies using apoD(M93-A) indicated M93 was required for HpETE reduction. We also assessed the impact that apoD MetSO formation has on protein aggregation by Western blotting of HpETE-treated apoD and human brain samples. ApoD Met oxidation was associated with formation of apoD aggregates that were also detected in AD hippocampus. In conclusion, conversion of HpETE to HETE is mediated by apoD M93, a process that may contribute to apoD antioxidant function.

Macromolecular and small molecule modulation of intracellular A{beta}42 aggregation and associated toxicity

2011-12-09T14:06:40Z

Aβ peptide has a central role in Alzheimer’s disease (AD) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aβ aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aβ peptide. Here we establish a mammalian cell system for the direct visualisation of β-amyloid formation by expression of an Aβ₄₂-EGFP fusion protein in the HEK cell line, T-REx293, and use this to identify both macromolecules and small molecules that reduced aggregation and associated cell toxicity. Thus, a molecular shield protein, AavLEA1, which limits aggregation of proteins with expanded polyglutamine (polyQ) repeats, is also effective against Aβ₄₂-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 and ganoderic acid DM, respectively, with activity against Aβ peptide. Both PS5 and ganoderic acid DM probably promote Aβ aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aβ peptide on cell physiology, and to identify reagents that counteract those effects.

Reprogramming of pancreatic exocrine cells towards a beta cell character using Pdx1, Ngn3 and MafA

E Akinci, A Banga, L V Greder, J R Dutton, J M.W. Slack — 2011-12-09T12:09:52Z

Pdx1, Ngn3 and MafA have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta cells in vivo (Zhou et al. (2008) Nature 455, 627-630), We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM. When introduced into AR42j-B13 cells, Ad-PNM causes a rapid change to a flattened morphology, and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are upregulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 and Ins2 gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.

Oligoamine Analogues in Combination with 2-Difluoromethylornithine (DFMO) Synergistically Induce Re-expression of Aberrantly Silenced Tumor Suppressor Genes

Y Wu, N Steinbergs, T Murray-Stewart, L J. Marton, R A Casero Jr. — 2011-12-02T10:55:23Z

Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumor suppressor genes. Lysine-specific demethylase 1 (LSD1) was the first enzyme identified to specifically demethylate lysine 4 of histone H3 (H3K4). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide binding, amine oxidase domain of LSD1 is homologous to two polyamine oxidases, spermine oxidase and N¹-acetylpolyamine oxidase. We have demonstrated that long chain polyamine analogues, the oligoamines, are inhibitors of LSD1. Here we report the synergistic effects of specific oligoamines in combination with 2-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumor suppressor genes, including the secreted frizzled-related protein 2 (SFRP2) gene, which encodes a Wnt signaling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer.

Up-regulated miR-17 promotes cell proliferation, tumor growth and cell cycle progression by targeting RND3 tumor suppressor gene in colorectal carcinoma

H Luo, J Zou, Z Dong, Q Zeng, D Wu, L Liu — 2011-12-01T15:34:18Z

Emerging evidence indicates that the miR-17 family may have a causal role in human cancer tumorigenesis, but their specific effects on occurrence of colorectal carcinoma (CRC) are still poorly understood. In this study, we profiled CRC tissue samples by miRNA microarray and found that four members of the miR-17 family had higher expressions in CRC tissues than in normal tissues. This finding was further validated by qRT-PCR. Transfecting CRC cells with an inhibitor of miR-17 lowered their ability to proliferate and induced G₀/G₁ arrest. We also confirmed that miR-17 exerted this function by directly targeting RND3 in vitro and that the expression of miR-17 was negatively correlated with that of RND3 in CRC tissues and CRC cells. Moreover, miR-17 inhibition led to tumor growth suppression and up-regulation of RND3 expression in a nude mouse xenograft model. RND3 expression was found significantly lower in CRC tissues than in normal tissues and adenomas, indicating RND3 may act as a tumor suppressor gene in CRC. In conclusion, our study suggests that miR-17 plays an important role in CRC carcinogenesis by targeting RND3 and may be a therapeutic agent for CRC.

MOCA is an integrator of the neuronal death signals that are activated by familial Alzheimer{'}s disease-related mutants of amyloid beta precursor protein and presenilins

N Tachi, Y Hashimoto, M Matsuoka — 2011-11-24T15:29:46Z

Death in cholinergic neurons in cerebral cortex and certain subcortical regions is linked to irreversible dementia relevant to Alzheimer’s disease (AD). Although multiple studies have shown that expression of a familial AD (FAD)-linked amyloid beta precursor protein (APP) or a presenilin (PS) mutant, but not that of wild-type APP or PS, induced neuronal death by activating intracellular death signals, it remains to be addressed how these signals are interrelated and what the key molecule involving this process is. In this study, we show that the PS1-mediated (or possibly the PS2-mediated) signal is essential for the APP-mediated death in a gamma-secretase-independent manner and vice versa. MOCA (modifier of cell adhesion), originally identified as being a PS- and Rac1-binding protein, is a common downstream constituent of these neuronal death signals. The detailed molecular analysis indicates that MOCA is a key molecule of the AD-relevant neuronal death signals that links the PS-mediated death signal with the APP-mediated death signal at a point between Rac1 (or Cdc42) and ASK1.

Oxidation of Met1606 in von Willebrand Factor is a Risk Factor for Thrombotic and Septic Complications in Chronic Renal Failure

2011-11-18T11:49:19Z

Chronic Kidney Disease (CKD) is a life-threatening pathology, often requiring haemodialysis (HD) and characterized by high oxidative stress, inflammation and perturbation of vascular endothelium. HD-patients have increased levels of von Willebrand factor (vWF), a large protein (~240kDa) released as ultra large polymers (UL-vWF, M.W.~20000-50000kDa) from vascular endothelial cells and megakaryocytes and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13, a zinc-protease cleaving vWF at the single Tyr1605-Met1606 bond, and by leukocyte serine proteases, released by activated polymorphonuclear cells during bacterial infections. Previous studies showed that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF, which are not only more prothrombotic than shorter vWF oligomers but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD-patients have greatly increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In this study, we first purified vWF from HD-patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers, which are also more resistant to ADAMTS-13. Using targeted mass spectrometry techniques, we estimated that HD-vWF contains <10% of Met1606 in the sulphoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate more efficiently platelets and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.

Mesothelin Enhances Invasion of Ovarian Cancer by Inducing MMP-7 through MAPK/ERK and JNK Pathways

M Chang, C Chen, P Chen, Y Chiang, Y Chen, T Mao, H Lin, W Lin Chiang, W Cheng — 2011-10-14T13:22:35Z

Ovarian cancer has one of the highest mortalities in malignancies in women but little is known of its tumor progression properties and there is still no effective molecule that can monitor its growth or therapeutic responses. Mesothelin, a secreted protein that is over-expressed in ovarian cancer tissues with a poor clinical outcome, has been previously identified to activate PI3K/Akt signaling and inhibit paclitaxel-induced apoptosis. This study investigated the correlation between mesothelin and matrix metalloproteinases-7 (MMP-7) in the progression of ovarian cancer, and mesothelin’s mechanism of enhancing ovarian cancer invasion. The expression of mesothelin correlated well with MMP-7 expression in human ovarian cancer tissues. Mesothelin over-expressed or mesothelin-treated ovarian cancer cells enhanced the migration and invasion of cancer cells through the inducation of MMP-7. Mesothelin regulated the expression of MMP-7 through the ERK (extracellular-signal-regulated kinase) 1/2, Akt (phosphoinositide 3-kinase), and JNK (c-jun-N-terminal kinase) pathways. The expression of MMP-7 and the migrating ability of mesothelin-treated ovarian cancer cells were suppressed by the ERK1/2 or JNK specific inhibitor, or decoy AP-1 oligonucleotide by in vitro experiments, while in vivo animal experiments also demonstrated that mice treated with MAPK/ERK or JNK-specific inhibitors could decrease intra-tumor MMP-7 expression, delay tumor growth, and extend the survival of the mice. In conclusion, mesothelin enhances ovarian cancer invasion by MMP-7 expression through the MAPK/ERK and JNK signal transduction pathways. Blocking the mesothelin-related pathway can be a potential strategy for inhibiting the growth of ovarian cancer.