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  • "Omics" of human sperm: profiling protein phosphatases. 23895272

    Phosphorylation is a major regulatory mechanism in eukaryotic cells performed by the concerted actions of kinases and phosphatases (PPs). Protein phosphorylation has long been relevant to sperm physiology, from acquisition of motility in the epididymis to capacitation in the female reproductive tract. While the precise kinases involved in the regulation of sperm phosphorylation have been studied for decades, the PPs have only recently received research interest. Tyrosine phosphorylation was first implicated in the regulation of several sperm-related functions, from capacitation to oocyte binding. Only afterwards, in 1996, the inhibition of the serine/threonine-PP phosphoprotein phosphatase 1 (PPP1) by okadaic acid and calyculin-A was shown to initiate motility in caput epididymal sperm. Today, the current mechanisms of sperm motility acquisition based on PPP1 and its regulators are still far from being fully understood. PPP1CC2, specifically expressed in mammalian sperm, has been considered to be the only sperm-specific serine/threonine-PP, while other PPP1 isoforms were thought to be absent from sperm. This article examines the "Omics" of human sperm, and reports, for the first time, the identification of three new serine/threonine-protein PPs, PPP1CB, PPP4C, and PPP6C, in human sperm, together with two tyrosine-PPs, MKP1 and PTP1C. We specifically localized in sperm PPP1CB and PPP1CC2 from the PPP1 subfamily, and PPP2CA, PPP4C, and PPP6C from the PPP2 subfamily of the serine/threonine-PPs. A semi-quantitative analysis was performed to determine the various PPs' differential expression in sperm head and tail. These findings contribute to a comprehensive understanding of human sperm PPs, and warrant further research for their clinical and therapeutic significance.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • α7 nicotinic acetylcholine receptor agonist PNU-282987 attenuates early brain injury in a perforation model of subarachnoid hemorrhage in rats. 21960575

    Early brain injury is an important pathological process after subarachnoid hemorrhage (SAH). The goal of this study was to evaluate whether the α7 nicotinic acetylcholine receptor (α7nAChR) agonist PNU-282987 attenuates early brain injury after SAH and whether α7nAChR stimulation is associated with down-regulation of caspase activity via phosphatidylinositol 3-kinase-Akt signaling.The perforation model of SAH was performed, and neurological score, body weight loss, and brain water content were evaluated 24 and 72 hours after surgery. Western blot and immunohistochemistry were used for quantification and localization of phosphorylated Akt and cleaved caspase 3. Neuronal cell death was quantified with TUNEL staining. α7nAChR antagonist methylcaconitine and phosphatidylinositol 3-kinase inhibitor wortmannin were used to manipulate the proposed pathway, and results were quantified with Western blot.PNU-282987 improved neurological deficits both 24 and 72 hours after surgery and reduced brain water content in left hemispheres 24 hours after surgery. PNU-282987 significantly increased phosphorylated Akt levels and significantly decreased cleaved caspase 3 levels in ipsilateral hemispheres after SAH. Methylcaconitine and wortmannin reversed effects of treatment. Phosphorylated Akt and cleaved caspase 3 were colocalized to neurons in the ipsilateral basal cortex. Phosphorylated Akt was mainly localized in TUNEL-negative cells. PNU-282987 significantly reduced neuronal cell death in the ipsilateral basal cortex.α7nAChR stimulation decreased neuronal cell death and brain edema and improved neurological status in a rat perforation model of SAH. α7nAChR stimulation is associated with increasing phosphorylation of Akt and decreasing cleaved caspase 3 levels in neurons.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60
  • 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-Estradiol unmasks metabotropic receptor-mediated metaplasticity of NMDA receptor transmission in the female rat dentate gyrus. 22541715

    Loss of estrogen in women following menopause is associated with increased risk for cognitive decline, dementia and depression, all of which can be prevented by estradiol replacement. The dentate gyrus plays an important role in cognition, learning and memory. The gatekeeping function of the dentate gyrus to filter incoming activity into the hippocampus is modulated by estradiol in a frequency-dependent manner and involves activation of metabotropic glutamate receptors (mGluR). In the present study, we investigated whether estradiol (EB) modulates the metaplastic effect of inducing synaptic long-term potentiation (LTP) on subsequent propensity for expression of LTP in the dentate gyrus. At medial perforant path-dentate granule cell synapses in hippocampal slices of ovariectomized female rats, EB replacement was critical for an initial induction of LTP to enhance the magnitude of subsequent LTP elicited by a second high-frequency stimulation, metaplasticity, which was not present in slices from oil-treated control animals. EB enhanced expression of group I mGluRs, and the metaplastic effect of EB on LTP required activation of group I mGluRs that led to Src-family tyrosine kinase-mediated phosphorylation of NR2B subunits of N-methyl-d-aspartate receptors (NMDAR) that enhanced the magnitude of NMDAR-dependent LTP. Our data show that EB effects on LTP in the hippocampal dentate gyrus require activation of group I mGluRs, which in turn leads to functional metaplastic regulation of NR2B subunit-containing NMDARs, as opposed to direct effects of EB on NMDARs.
    Document Type:
    Reference
    Product Catalog Number:
    AB5675
    Product Catalog Name:
    Anti-Metabotropic Glutamate Receptor 5 Antibody, pain
  • β Integrins mediate FAK Y397 autophosphorylation of resistance arteries during eutrophic inward remodeling in hypertension. 25300309

    Human essential hypertension is characterized by eutrophic inward remodeling of the resistance arteries with little evidence of hypertrophy. Upregulation of αVβ3 integrin is crucial during this process. In order to investigate the role of focal adhesion kinase (FAK) activation in this process, the level of FAK Y397 autophosphorylation was studied in small blood vessels from young TGR(mRen2)27 animals as blood pressure rose and eutrophic inward remodeling took place. Between weeks 4 and 5, this process was completed and accompanied by a significant increase in FAK phosphorylation compared with normotensive control animals. Phosphorylated (p)FAK Y397 was coimmunoprecipitated with both β1- and β3-integrin-specific antibodies. In contrast, only a fraction (less than 10-fold) was coprecipitated with the β3 integrin subunit in control vessels. Inhibition of eutrophic remodeling by cRGDfV treatment of TGR(mRen2)27 rats resulted in the development of smooth-muscle-cell hypertrophy and a significant further enhancement of FAK Y397 phosphorylation, but this time with exclusive coassociation of pFAK Y397 with integrin β1. We established that phosphorylation of FAK Y397 with association with β1 and β3 integrins occurs with pressure-induced eutrophic remodeling. Inhibiting this process leads to an adaptive hypertrophic vascular response induced by a distinct β1-mediated FAK phosphorylation pattern.
    Document Type:
    Reference
    Product Catalog Number:
    AB1952
    Product Catalog Name:
    Anti-Integrin beta1 Antibody, Cytosolic
  • β1 integrin gene excision in the adult murine cardiac myocyte causes defective mechanical and signaling responses. 22248583

    How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which β1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of β1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal-regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte β1 integrin expression modified adrenergic-mediated signaling through extracellular signal-regulated kinase, p38, and Akt. Reduction of β1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that β1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • β2 integrin induces TCRζ-Syk-phospholipase C-γ phosphorylation and paxillin-dependent granule polarization in human NK cells. 21270398

    Cytotoxic lymphocytes kill target cells through polarized release of the content of lytic granules at the immunological synapse. In human NK cells, signals for granule polarization and for degranulation can be uncoupled: Binding of β(2) integrin LFA-1 to ICAM is sufficient to induce polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. In this study, we investigated the basis for this difference. IL-2-expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR ζ-chain, tyrosine kinase Syk, and phospholipase C-γ. Whereas CD16 activated Ca(2+) mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, phospholipase C-γ, and protein kinase C, as well as by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca(2+)-dependent degranulation and paxillin-dependent granule polarization.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • βCaMKII plays a nonenzymatic role in hippocampal synaptic plasticity and learning by targeting αCaMKII to synapses. 21752990

    The calcium/calmodulin-dependent kinase type II (CaMKII) holoenzyme of the forebrain predominantly consists of heteromeric complexes of the αCaMKII and βCaMKII isoforms. Yet, in contrast to αCaMKII, the role of βCaMKII in hippocampal synaptic plasticity and learning has not been investigated. Here, we compare two targeted Camk2b mouse mutants to study the role of βCaMKII in hippocampal function. Using a Camk2b(-/-) mutant, in which βCaMKII is absent, we show that both hippocampal-dependent learning and Schaffer collateral-CA1 long-term potentiation (LTP) are highly dependent upon the presence of βCaMKII. We further show that βCaMKII is required for proper targeting of αCaMKII to the synapse, indicating that βCaMKII regulates the distribution of αCaMKII between the synaptic pool and the adjacent dendritic shaft. In contrast, localization of αCaMKII, hippocampal synaptic plasticity and learning were unaffected in the Camk2b(A303R) mutant, in which the calcium/calmodulin-dependent activation of βCaMKII is prevented, while the F-actin binding and bundling property is preserved. This indicates that the calcium/calmodulin-dependent kinase activity of βCaMKII is fully dispensable for hippocampal learning, LTP, and targeting of αCaMKII, but implies a critical role for the F-actin binding and bundling properties of βCaMKII in synaptic function. Together, our data provide compelling support for a model of CaMKII function in which αCaMKII and βCaMKII act in concert, but with distinct functions, to regulate hippocampal synaptic plasticity and learning.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • γ-Catenin at adherens junctions: mechanism and biologic implications in hepatocellular cancer after β-catenin knockdown. 23555187

    β-Catenin is important in liver homeostasis as a part of Wnt signaling and adherens junctions (AJs), while its aberrant activation is observed in hepatocellular carcinoma (HCC). We have reported hepatocyte-specific β-catenin knockout (KO) mice to lack adhesive defects as γ-catenin compensated at AJ. Because γ-catenin is a desmosomal protein, we asked if its increase in KO might deregulate desmosomes. No changes in desmosomal proteins or ultrastructure other than increased plakophilin-3 were observed. To further elucidate the role and regulation of γ-catenin, we contemplate an in vitro model and show γ-catenin increase in HCC cells upon β-catenin knockdown (KD). Here, γ-catenin is unable to rescue β-catenin/T cell factor (TCF) reporter activity; however, it sufficiently compensates at AJs as assessed by scratch wound assay, centrifugal assay for cell adhesion (CAFCA), and hanging drop assays. γ-Catenin increase is observed only after β-catenin protein decrease and not after blockade of its transactivation. γ-Catenin increase is associated with enhanced serine/threonine phosphorylation and abrogated by protein kinase A (PKA) inhibition. In fact, several PKA-binding sites were detected in γ-catenin by in silico analysis. Intriguingly γ-catenin KD led to increased β-catenin levels and transactivation. Thus, γ-catenin compensates for β-catenin loss at AJ without affecting desmosomes but is unable to fulfill functions in Wnt signaling. γ-Catenin stabilization after β-catenin loss is brought about by PKA. Catenin-sensing mechanism may depend on absolute β-catenin levels and not its activity. Anti-β-catenin therapies for HCC affecting total β-catenin may target aberrant Wnt signaling without negatively impacting intercellular adhesion, provided mechanisms leading to γ-catenin stabilization are spared.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4
  • γH2A is a component of yeast heterochromatin required for telomere elongation. 21212735

    Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (γH2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that γH2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where γH2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of γH2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of γH2A at heterochromatin. To address the function of γH2A, we used a Δrif1 mutant strain in which telomeres are excessively elongated to show that γH2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.
    Document Type:
    Reference
    Product Catalog Number:
    05-419
    Product Catalog Name:
    Anti-Myc Tag Antibody, clone 9E10