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  • Troponin I is present in human cartilage and inhibits angiogenesis. 10077564

    Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1691
    Product Catalog Name:
    Anti-Troponin I Antibody, a.a. 186-192, clone C5

  • Mutagenesis and analysis of genetic mutations in the GC-rich KISS1 receptor sequence identified in humans with reproductive disorders. 21912371

    The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood (1,2,3). Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) (1,2) and precocious puberty (4). Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR. Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well. The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies (1,4,5,6,7,8). As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation (4) as well as to alter the rate of degradation of KISS1R (9). Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors (9). In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.
    Document Type:
    Reference
    Product Catalog Number:
    16-219
    Product Catalog Name:
    Anti-Myc Tag Antibody, clone 4A6, agarose conjugate

  • Ischemia induces closure of gap junctional channels and opening of hemichannels in heart-derived cells and tissue. 21865853

    Gap junction intercellular communication (GJIC) and hemichannel permeability may have important roles during an ischemic insult. Our aim was to evaluate the effect of ischemia on gap junction channels and hemichannels.We used neonatal rat heart myofibroblasts and simulated ischemia with a HEPES buffer with high potassium, low pH, absence of glucose, and oxygen tension was reduced by dithionite. Microinjection, western blot, immunofluorescence, cell viability and dye uptake were used to evaluate the effects induced by dithionite. Isolated perfused rat hearts were used to analyse infarct size.Short period with simulated ischemia reduced the ability to transfer a dye between neighbouring cells, which indicated reduced GJIC. Prolonged exposure to simulated ischemia caused opening of hemichannels, and cell death was apparent while gap junction channels remained closed. Connexin 43 became partially dephosphorylated and the total amount decreased during simulated ischemia. We were not able to detect the alternative hemichannel-forming protein, Pannexin 1, in these cells. The potential importance of Connexin 43 or Pannexin 1 hemichannels in ischemia-induced infarct in the intact heart was studied by perfusion of the heart in the presence of peptides that block one or the other type of hemichannels. The connexin-derived peptide, Gap26, significantly reduced the infract/risk zone ratio (control 48.7±4.2% and Gap26 19.4±4.1%, pless than 0.001), while the pannexin-derived peptide, (10)Panx1, did not change infarct/risk ratio.Connexin 43 is most likely responsible for both closure of gap junction channels and opening of hemichannels during simulated ischemia in neonatal rat heart myofibroblasts. Opening of connexin 43 hemichannels during ischemia-reperfusion seems to be an important mechanism for ischemia-reperfusion injury in the heart. By preventing the opening of these channels during early ischemia-reperfusion the infarct size becomes significantly reduced.
    Document Type:
    Reference
    Product Catalog Number:
    AB3841
    Product Catalog Name:
    Anti-Connexin 43 Antibody, phospho-specific (Ser368)

  • Essential role of DPPA3 for chromatin condensation in mouse oocytogenesis. 22034526

    Dynamic alterations in chromatin configuration occur in mammalian oocytogenesis. Based on chromatin configuration patterns, fully grown oocytes are classified into two types. One is surrounded nucleolus (SN)-type and the other is nonsurrounded nucleolus (NSN)-type oocytes. Although chromatin condensation during the transition from NSN- to SN-type oocytes is a prerequisite for normal early embryonic development, the molecular mechanisms remain unclear. In this study, we analyzed the role of DPPA3 (also known as PGC7/Stella) in this transition using Dppa3-null oocytes. The NSN-to-SN transition was significantly impaired, and transcriptional repression was incomplete in the Dppa3-null oocytes. Additionally, we revealed that prior transcriptional repression was necessary for the NSN-to-SN transition. These findings demonstrate that DPPA3 is an essential factor for the production of functional oocytes through transcriptional repression and chromatin condensation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Dopamine-dependent tuning of striatal inhibitory synaptogenesis. 20181591

    Dopaminergic projections to the striatum, crucial for the correct functioning of this brain region in adulthood, are known to be established early in development, but their role is currently uncharacterized. We demonstrate here that dopamine, by activating D(1)- and/or D(2)-dopamine receptors, decreases the number of functional GABAergic synapses formed between the embryonic precursors of the medium spiny neurons, the principal output neurons of the striatum, with associated changes in spontaneous synaptic activity. Activation of these receptors reduces the size of postsynaptic GABA(A) receptor clusters and their overall cell-surface expression, without affecting the total number of clusters or the size or number of GABAergic nerve terminals. These changes result from an increased internalization of GABA(A) receptors, and are mediated by distinct signaling pathways converging at the level of GABA(A) receptors to cause a transient PP2A/PP1-dependent dephosphorylation. Thus, tonic D(1)- and D(2)-receptor activity limits the extent of collateral inhibitory synaptogenesis between medium spiny neurons, revealing a novel role of dopamine in controlling the development of intrinsic striatal microcircuits.
    Document Type:
    Reference
    Product Catalog Number:
    MAB341
    Product Catalog Name:
    Anti-GABA A Receptor β 2,3 Chain Antibody, clone BD17

  • The Mannich base NC1153 promotes long-term allograft survival and spares the recipient from multiple toxicities. 16177063

    JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-alpha-driven NF-kappaB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Striatal enriched phosphatase 61 dephosphorylates Fyn at phosphotyrosine 420. 11983687

    A family of protein tyrosine phosphatases enriched within the central nervous system called striatal enriched phosphatase (STEP) has been implicated in the regulation of the N-methyl-d-aspartate receptor. STEP(61), a membrane-associated isoform located in the postsynaptic densities (PSDs) of striatal neurons, contains two transmembrane domains, two proline-rich domains, and a kinase-interacting motif. This study demonstrates that STEP(61) associates with Fyn, a member of the Src family kinases that is also enriched in PSDs. By using human embryonic kidney 293 cells for co-transfection, we determined that a substrate-trapping variant (STEP(61) CS) binds to Fyn but not to other members of the Src family present in PSDs. In a complementary experiment, myc-tagged Fyn immunoprecipitates STEP(61) CS. STEP(61) binds to Fyn through one of its proline-rich domains and the kinase-interacting motif domain, whereas Fyn binds to STEP(61) through its Src homology 2 domain and the unique N-terminal domain. STEP(61) CS pulls down Fyn when the Tyr(420) site is phosphorylated. In vitro, wild-type STEP(61) dephosphorylates Fyn at Tyr(420) but not at Tyr(531). These results suggest that STEP regulates the activity of Fyn by specifically dephosphorylating the regulatory Tyr(420) and may be one mechanism by which Fyn activity is decreased within PSDs.
    Document Type:
    Reference
    Product Catalog Number:
    LP1
    Product Catalog Name:
    VLDL, human

  • Loop variants of the serpin thyroxine-binding globulin: implications for hormone release upon limited proteolysis. 11931635

    Thyroxine-binding globulin (TBG) and corticosteroid-binding globulin are unique among non-inhibitory members of the superfamily of serine-proteinase inhibitors (serpins) in undergoing a dramatic increase in stability [stressed-to-relaxed (S-->R) transition] after proteolytic cleavage within their exposed reactive-site-loop (RSL) equivalent. This structural rearrangement involves the insertion of the cleaved loop as a new strand into the beta-sheet A and is accompanied by a decrease in hormone binding. To define the mechanism that leads to disruption of hormone binding of TBG after proteolytic cleavage, the effect of partial loop deletions and replacements by the alpha(1)-proteinase inhibitor homologues of TBG were evaluated. Unexpectedly, deletion of the loop's C-terminus, thought to be important for thyroxine binding, improved the binding affinity over that of normal TBG. Proteolytic cleavage of this variant revealed an intact S-->R transition and reduced its binding activity to that of cleaved TBG. In contrast, a chimaera with C-terminal loop extension mimicked the decreased binding affinity of cleaved TBG and had a thermal stability intermediate between that of native and cleaved serpins. This variant was still susceptible to loop cleavage and underwent an S-->R transition, yet without changing its binding affinity. Our data exclude a direct involvement of loop residues in thyroxine binding of native TBG. Limited insertion of the RSL into beta-sheet A appears to trigger hormone release after proteolytic cleavage. In support of this concept, residues within the hinge region of the TBG loop are phylogenetically highly conserved, suggestive of their physiological role as a functional switch in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    05-100

  • Withdrawal from cocaine self-administration alters NMDA receptor-mediated Ca2+ entry in nucleus accumbens dendritic spines. 22870207

    We previously showed that the time-dependent intensification ("incubation") of cue-induced cocaine seeking after withdrawal from extended-access cocaine self-administration is accompanied by accumulation of Ca(2+)-permeable AMPA receptors (CP-AMPARs) in the rat nucleus accumbens (NAc). These results suggest an enduring change in Ca(2+) signaling in NAc dendritic spines. The purpose of the present study was to determine if Ca(2+) signaling via NMDA receptors (NMDARs) is also altered after incubation. Rats self-administered cocaine or saline for 10 days (6 h/day). After 45-47 days of withdrawal, NMDAR-mediated Ca(2+) entry elicited by glutamate uncaging was monitored in individual NAc dendritic spines. NMDAR currents were simultaneously recorded using whole cell patch clamp recordings. We also measured NMDAR subunit levels in a postsynaptic density (PSD) fraction prepared from the NAc of identically treated rats. NMDAR currents did not differ between groups, but a smaller percentage of spines in the cocaine group responded to glutamate uncaging with NMDAR-mediated Ca(2+) entry. No significant group differences in NMDAR subunit protein levels were found. The decrease in the proportion of spines showing NMDAR-mediated Ca(2+) entry suggests that NAc neurons in the cocaine group contain more spines which lack NMDARs (non-responding spines). The fact that cocaine and saline groups did not differ in NMDAR currents or NMDAR subunit levels suggests that the number of NMDARs on responding spines is not significantly altered by cocaine exposure. These findings are discussed in light of increases in dendritic spine density in the NAc observed after withdrawal from repeated cocaine exposure.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple