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  • α-Actinin-4 enhances colorectal cancer cell invasion by suppressing focal adhesion maturation. 25860875

    α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4
  • α-Synuclein-induced synapse damage in cultured neurons is mediated by cholesterol-sensitive activation of cytoplasmic phospholipase A2. 25761116

    The accumulation of aggregated forms of the α-synuclein (αSN) is associated with the pathogenesis of Parkinson's disease (PD) and Dementia with Lewy Bodies. The loss of synapses is an important event in the pathogenesis of these diseases. Here we show that aggregated recombinant human αSN, but not βSN, triggered synapse damage in cultured neurons as measured by the loss of synaptic proteins. Pre-treatment with the selective cytoplasmic phospholipase A2 (cPLA2) inhibitors AACOCF3 and MAFP protected neurons against αSN-induced synapse damage. Synapse damage was associated with the αSN-induced activation of synaptic cPLA2 and the production of prostaglandin E2. The activation of cPLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B or Hexa-PAF) also protect neurons against αSN-induced synapse damage. αSN-induced synapse damage was also reduced in neurons pre-treated with the cholesterol synthesis inhibitor (squalestatin). These results are consistent with the hypothesis that αSN triggered synapse damage via hyperactivation of cPLA2. They also indicate that αSN-induced activation of cPLA2 is influenced by the cholesterol content of membranes. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse damage seen during PD.
    Document Type:
    Reference
    Product Catalog Number:
    MAB368
  • α-Synuclein accumulates in huntingtin inclusions but forms independent filaments and its deficiency attenuates early phenotype in a mouse model of Huntington's disease. 22045698

    Huntington's disease (HD) is the most common of nine inherited neurological disorders caused by expanded polyglutamine (polyQ) sequences which confer propensity to self-aggregate and toxicity to their corresponding mutant proteins. It has been postulated that polyQ expression compromises the folding capacity of the cell which might affect other misfolding-prone proteins. α-Synuclein (α-syn) is a small neural-specific protein with propensity to self-aggregate that forms Parkinson's disease (PD) Lewy bodies. Point mutations in α-syn that favor self-aggregation or α-syn gene duplications lead to familial PD, thus indicating that increased α-syn aggregation or levels are sufficient to induce neurodegeneration. Since polyQ inclusions in HD and other polyQ disorders are immunopositive for α-syn, we speculated that α-syn might be recruited as an additional mediator of polyQ toxicity. Here, we confirm in HD postmortem brains and in the R6/1 mouse model of HD the accumulation of α-syn in polyQ inclusions. By isolating the characteristic filaments formed by aggregation-prone proteins, we found that N-terminal mutant huntingtin (N-mutHtt) and α-syn form independent filamentous microaggregates in R6/1 mouse brain as well as in the inducible HD94 mouse model and that N-mutHtt expression increases the load of α-syn filaments. Accordingly, α-syn knockout results in a diminished number of N-mutHtt inclusions in transfected neurons and also in vivo in the brain of HD mice. Finally, α-syn knockout attenuates body weight loss and early motor phenotype of HD mice. This study therefore demonstrates that α-syn is a modifier of polyQ toxicity in vivo and raises the possibility that potential PD-related therapies aimed to counteract α-syn toxicity might help to slow HD.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5374
    Product Catalog Name:
    Anti-Huntingtin Protein Antibody, clone mEM48
  • α-synuclein induced synapse damage is enhanced by amyloid-β1-42. 21138585

    The pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) is associated with the accumulation of aggregated forms of the α-synuclein (αSN) protein. An early event in the neuropathology of PD and DLB is the loss of synapses and a corresponding reduction in the level of synaptic proteins. However, the molecular mechanisms involved in synapse damage in these diseases are poorly understood. In this study the process of synapse damage was investigated by measuring the amount of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission, in cultured neurons incubated with αSN, or with amyloid-β (Aβ) peptides that are thought to trigger synapse degeneration in Alzheimer's disease.We report that the addition of recombinant human αSN reduced the amount of synaptophysin in cultured cortical and hippocampal neurons indicative of synapse damage. αSN also reduced synaptic vesicle recycling, as measured by the uptake of the fluorescent dye FM1-43. These effects of αSN on synapses were modified by interactions with other proteins. Thus, the addition of βSN reduced the effects of αSN on synapses. In contrast, the addition of amyloid-β (Aβ)1-42 exacerbated the effects of αSN on synaptic vesicle recycling and synapse damage. Similarly, the addition of αSN increased synapse damage induced by Aβ1-42. However, this effect of αSN was selective as it did not affect synapse damage induced by the prion-derived peptide PrP82-146.These results are consistent with the hypothesis that oligomers of αSN trigger synapse damage in the brains of Parkinson's disease patients. Moreover, they suggest that the effect of αSN on synapses may be influenced by interactions with other peptides produced within the brain.
    Document Type:
    Reference
    Product Catalog Number:
    MAB368
  • α-Synuclein levels modulate Huntington's disease in mice. 22010050

    α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinson's disease (PD) and Huntington's disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • α(V)β(3) integrin-targeted PLGA-PEG nanoparticles for enhanced anti-tumor efficacy of a Pt(IV) prodrug. 22584163

    Targeted delivery of therapeutics to tumor neovasculature is potentially a powerful approach for selective cancer treatment. Integrins are heterodimeric transmembrane proteins involved in cell adhesion and cell signaling, and their expression is commonly upregulated in cancers and inflammatory diseases. The α(v)β(3) integrin is differentially upregulated on angiogenic endothelial cells as well as on many cancer cells. Here we demonstrate the differential targeting of cisplatin prodrug-encapsulated poly(d,l-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) to the α(v)β(3) integrin on cancer cells using the cyclic pentapeptide c(RGDfK). Cisplatin is one of the most widely used anticancer drugs, and approaches that can improve its therapeutic index are of broad importance. The RGD-targeted Pt(IV)-encapsulated NPs displayed enhanced cytotoxicity as compared to cisplatin administered in its conventional dosage form in model prostate and breast cancer epithelial cells in vitro. Cytotoxicities were also elevated in comparison to those of previously reported systems, a small molecule Pt(IV)-RGD conjugate and a Pt(IV) nanoscale coordination polymer carrying RGD moieties. This result encouraged us also to evaluate the anticancer effect of the new construct in an animal model. The RGD-targeted PLGA-PEG NPs were more efficacious and better tolerated by comparison to cisplatin in an orthotopic human breast cancer xenograft model in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1976F
    Product Catalog Name:
    Anti-Integrin αVβ3 Antibody, clone LM609, FITC conjugated
  • β-adrenergic receptor blockade reduces endoplasmic reticulum stress and normalizes calcium handling in a coronary embolization model of heart failure in canines. 21493701

    Alterations in calcium homeostasis in the endoplasmic/sarcoplasmic reticulum (ER) cause ER stress that ultimately may affect ventricular function. However, the role of ER stress in β-blocker therapy for congestive heart failure (CHF) has not been studied. This study examined ER stress in CHF and evaluated its role in β-blocker therapy in a canine model of ischaemic CHF.CHF was created by daily coronary embolization in chronically instrumented dogs. After oral administration of β-blocker metoprolol or vehicle for 12 weeks, Ca(2+) transport proteins including sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), ryanodine receptor (RyR2), Na(+)-Ca(2+) exchanger (NCX1), Ca(2+) storage protein calreticulin (CRT), and phospholamban were evaluated by Western blot analysis. Cellular levels of ER stress marker, phosphorylated eukaryotic initiation factor 2α (eIF2α-P), were also examined. Compared with the vehicle-treated group, metoprolol caused significantly improved cardiac function, restored the proteins of SERCA2a, NCX1, and CRT, increased phosphorylated phospholamban, reversed protein kinase A hyperphosphorylation of RyR2, and resulted in normalized ER stress marker eIF2α-P and reduced DNA damage.Our results suggest that ER stress could be induced by abnormal Ca(2+) homeostasis in CHF. The restoration of calcium-handling protein function and resultant decrease in ER stress might, in part, explain the beneficial effects of β-blockade observed in CHF. Whether this mechanism occurs in other animal CHF models or human CHF warrants further study.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • β-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3. 22371497

    O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.
    Document Type:
    Reference
    Product Catalog Number:
    06-570
    Product Catalog Name:
    Anti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker
  • β₂-glycoprotein I: a novel component of innate immunity. 21454452

    Sepsis is a systemic host response to invasive infection by bacteria. Despite treatment with antibiotics, current mortality rates are in the range of 20%-25%, which makes sepsis the most important cause of death in intensive care. Gram-negative bacteria are a prominent cause of sepsis. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, plays a major role in activating the host's immune response by binding to monocytes and other cells. Several proteins are involved in neutralization and clearance of LPS from the bloodstream. Here, we provide evidence that β₂-glycoprotein I (β₂GPI) is a scavenger of LPS. In vitro, β₂GPI inhibited LPS-induced expression of tissue factor and IL-6 from monocytes and endothelial cells. Binding of β₂GPI to LPS caused a conformational change in β₂GPI that led to binding of the β₂GPI-LPS complex to monocytes and ultimately clearance of this complex. Furthermore, plasma levels of β₂GPI were inversely correlated with temperature rise and the response of inflammatory markers after a bolus injection of LPS in healthy individuals. Together, these observations provide evidence that β₂GPI is involved in the neutralization and clearance of LPS and identify β₂GPI as a component of innate immunity.
    Document Type:
    Reference
    Product Catalog Number:
    UFC30GV0S
    Product Catalog Name:
    Ultrafree-MC Centrifugal Filter