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  • C7 is expressed on endothelial cells as a trap for the assembling terminal complement complex and may exert anti-inflammatory function. 19179470

    We describe a novel localization of C7 as a membrane-bound molecule on endothelial cells (ECs). Data obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, Northern blot analysis, and mass spectrometry revealed that membrane-associated C7 (mC7) was indistinguishable from soluble C7 and was associated with vimentin on the cell surface. mC7 interacted with the other late complement components to form membrane-bound TCC (mTCC). Unlike the soluble SC5b-9, mTCC failed to stimulate ECs to express adhesion molecules, to secrete IL-8, and to induce albumin leakage through a monolayer of ECs, and more importantly protected ECs from the proinflammatory effect of SC5b-9. Our data disclose the possibility of a novel role of mC7 that acts as a trap for the late complement components to control excessive inflammation induced by SC5b-9.
    Document Type:
    Reference
    Product Catalog Number:
    CBL180

  • CalDAG-GEFI down-regulation in the striatum as a neuroprotective change in Huntington's disease. 20147317

    Huntingtin protein (Htt) is ubiquitously expressed, yet Huntington's disease (HD), a fatal neurologic disorder produced by expansion of an Htt polyglutamine tract, is characterized by neurodegeneration that occurs primarily in the striatum and cerebral cortex. Such discrepancies between sites of expression and pathology occur in multiple neurodegenerative disorders associated with expanded polyglutamine tracts. One possible reason is that disease-modifying factors are tissue-specific. Here, we show that the striatum-enriched protein, CalDAG-GEFI, is severely down-regulated in the striatum of mouse HD models and is down-regulated in HD individuals. In the R6/2 transgenic mouse model of HD, striatal neurons with the largest aggregates of mutant Htt have the lowest levels of CalDAG-GEFI. In a brain-slice explant model of HD, knock-down of CalDAG-GEFI expression rescues striatal neurons from pathology induced by transfection of polyglutamine-expanded Htt exon 1. These findings suggest that the striking down-regulation of CalDAG-GEFI in HD could be a protective mechanism that mitigates Htt-induced degeneration.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5374
    Product Catalog Name:
    Anti-Huntingtin Protein Antibody, clone mEM48

  • Calmodulin controls synaptic strength via presynaptic activation of calmodulin kinase II. 20237283

    Calmodulin regulates multifarious cellular processes via a panoply of target interactions. However, the central role, multiple isoforms, and complex target interactions of calmodulin make it difficult to examine its precise functions. Here, we analyzed calmodulin function in neurons using lentivirally delivered short-hairpin RNAs that suppressed expression of all calmodulin isoforms by approximately 70%. Calmodulin knockdown did not significantly alter neuronal survival or synapse formation but depressed spontaneous neuronal network activity. Strikingly, calmodulin knockdown decreased the presynaptic release probability almost twofold, without altering the presynaptic readily-releasable vesicle pool or postsynaptic neurotransmitter reception. In calmodulin knockdown neurons, presynaptic release was restored to wild-type levels by expression of constitutively active calmodulin-dependent kinase-IIalpha (CaMKIIalpha); in contrast, in control neurons, expression of constitutively active CaMKIIalpha had no effect on presynaptic release. Viewed together, these data suggest that calmodulin performs a major function in boosting synaptic strength via direct activation of presynaptic calmodulin-dependent kinase II.
    Document Type:
    Reference
    Product Catalog Number:
    05-173
    Product Catalog Name:
    Anti-Calmodulin Antibody

  • Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+ -binding protein. 21590275

    Calreticulin (CRT)-1 is a major Ca(2+)-buffering protein in the lumen of the endoplasmic reticulum. Human and murine CRT-2 was isolated in 2002, but the subcellular localization and function is still unclear. Here, we studied the intracellular localization and function of CRT-2 with hemagglutinin-tagged (HA-) human CRT-2. Western blotting revealed HA-CRT-2 as a single band at 50 kDa. Using immunofluorescence microscopy of cultured fibroblasts and epithelial cells transfected with HA-CRT-2 cDNA, labeling for HA-CRT-2 was seen as a reticular network with a nuclear envelope pattern that colocalized with calnexin and protein disulfide isomerase. Immunoelectron microscopy confirmed that HA-CRT-2 was localized in the lumen of the endoplasmic reticulum. Stains-all staining, a method to detect Ca(2+)-binding proteins, could not stain the immunoprecipitate of HA-CRT-2, although HA-CRT-1 immunoprecipitate was stained blue. These results indicate that the molecular weight of the non-tagged CRT-2 on SDS-PAGE is 49 kDa, and that CRT-2, as well as CRT-1, is localized in the lumen of the endoplasmic reticulum, but that CRT-2 capacity for Ca(2+)-binding may be absent or much lower than that of CRT-1.
    Document Type:
    Reference
    Product Catalog Number:
    07-204

  • Calsperin is a testis-specific chaperone required for sperm fertility. 21131354

    Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3(-/-) males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.
    Document Type:
    Reference
    Product Catalog Number:
    07-158

  • Catheter-based induction of renal ischemia/reperfusion in swine: description of an experimental model. 25263203

    Several techniques to induce renal ischemia have been proposed: clamp, PVA particles, and catheter-balloon. We report the development of a controlled, single-insult model of unilateral renal ischemia/reperfusion (I/R) without contralateral nephrectomy, using a suitable model, the pig. This is a balloon-catheter-based model using a percutaneous, interventional radiology procedure. One angioplasty balloon-catheter was placed into the right renal artery and inflated for 120 min and reperfusion over 24 h. Serial serums were sampled from the inferior vena cava and urine was directly sampled from the bladder throughout the experiment, and both kidneys were excised after 24 h of reperfusion. Analyses of renal structure and function were performed by hematoxylin-eosin/periodic Acid-Schiff, serum creatinine (SCr), blood urea nitrogen (BUN), fractional excretion of ions, and glucose, SDS-PAGE analysis of urinary proteins, and serum neutrophil gelatinase-associated lipocalin (NGAL). Total nitrated protein was quantified to characterize oxidative stress. Acute tubular necrosis (ATN) was identified in every animal, but only two animals showed levels of SCr above 150% of baseline values. As expected, I/R increased SCr and BUN. Fractional sodium, potassium, chloride, and bicarbonate excretion were modulated during ischemia. Serum-nitrated proteins and NGAL had two profiles: decreased with ischemia and increased after reperfusion. This decline was associated with increased protein excretion during ischemia and early reperfusion. Altogether, these data show that the renal I/R model can be performed by percutaneous approach in the swine model. This is a suitable translational model to study new early renal ischemic biomarkers and pathophysiological mechanisms in renal ischemia.
    Document Type:
    Reference
    Product Catalog Number:
    06-284
    Product Catalog Name:
    Anti-Nitrotyrosine Antibody

  • CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes. 18481209

    The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2 ( -/- ) chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2 (-/-) chondrocytes, confirming a defect in ECM production. Ccn2 ( -/- ) chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated with decreased expression of alpha5 integrin. Moreover, CCN2 can bind to integrin alpha5beta1 in chondrocytes and can stimulate increased expression of integrin alpha5. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in Ccn2 ( -/- ) chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM production and integrin alpha5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin. 10821432

    A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines. The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells. Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities. Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.
    Document Type:
    Reference
    Product Catalog Number:
    06-262

  • Cell type-specific regulation of SL-1 and SL-2 genes. Induction of the SL-2 gene but not the SL-1 gene by human keratinocytes in response to cytokines and phorbolesters. 8349617

    The stromelysin-2 (SL-2) gene is transcriptionally active in normal human keratinocytes and encodes a secreted, catalytically competent but latent matrix metalloproteinase. Phorbolester induction resulted in the emergence of SL-2 (but not SL-1 transcripts), whereas the opposite was true for human mucosal fibroblasts. Expression of keratinocyte SL-2 was also induced by the two keratinocyte growth factors, transforming growth factor-alpha and epidermal growth factor, by the proinflammatory cytokine, tumor necrosis factor-alpha, but, somewhat surprisingly, not by interleukin-1 beta. The latent SL-2 proenzyme was isolated from 12-O-tetradecanoylphorbol-13-acetate-induced keratinocytes by immunoaffinity chromatography using a cross-reactive antibody raised against human SL-1. This procedure led to the recovery of a single M(r) 54,000 molecular species at a level of approximately 0.2 microgram/ml of culture medium. Amino-terminal sequencing identified the protein as SL-2 and verified the predicted signal sequence cleavage site. Conformational activation of latent SL-2 precursor by SDS gave rise to a full-length, uncleaved (M(r) 54,000) active form and at the same time exposed a cryptic thiol group. By contrast, organomercurial activation resulted in autolytic truncation of the molecule with loss of M(r) approximately 10,000 propeptide. SL-2 shared with (human fibroblast) SL-1 the ability to cleave casein, to "superactivate" fibroblast type procollagenase, and to form apparently binary, SDS-resistant complexes with tissue inhibitor of metalloproteinases-1.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3306
    Product Catalog Name:
    Anti-MMP-3 Antibody, cross reacts w/hMMP-10, clone 55-2A4