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  • Calcium transport sensitive to ruthenium red in cytochrome oxidase vesicles reconstituted with mitochondrial proteins. 1723412

    We describe a calcium transport that is sensitive to ruthenium red in liposomes reconstituted with mitochondrial extracts. This system is able to build an internally negative membrane potential, which allows the electrogenic influx of Ca2+ and Sr2+. Proteins with molecular weights higher than 35 kDa were incorporated to the vesicles, and enhanced the accumulation of the cation in an energy-dependent fashion.
    Document Type:
    Reference
    Product Catalog Number:
    70-600

  • Intrinsic and acquired forms of resistance against the anticancer ruthenium compound KP1019 [indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] (FFC14A). 15331656

    KP1019 [indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] (FFC14A) is a metal complex with promising anticancer activity. Since chemoresistance is a major obstacle in chemotherapy, this study investigated the influence of several drug resistance mechanisms on the anticancer activity of KP1019. Here we demonstrate that the cytotoxic effects of KP1019 are neither substantially hampered by overexpression of the drug resistance proteins multidrug resistance-related protein 1, breast cancer resistance protein, and lung resistance protein nor the transferrin receptor and only marginally by the cellular p53 status. In contrast, P-glycoprotein overexpression weakly but significantly (up to 2-fold) reduced KP1019 activity. P-glycoprotein-related resistance was based on reduced intracellular KP1019 accumulation and reversible by known P-glycoprotein modulators. KP1019 dose dependently inhibited ATPase activity of P-glycoprotein with a K(i) of approximately 31 microM. Furthermore, it potently blocked P-glycoprotein-mediated rhodamine 123 efflux under serum-free conditions (EC(50), approximately 8 microM), however, with reduced activity at increased serum concentrations (EC(50) at 10% serum, approximately 35 microM). Moreover, P-glycoprotein-mediated daunomycin resistance could only be marginally restored by KP1019 in serum-containing medium, also indicating an influence of serum proteins on the interaction between KP1019 and P-glycoprotein. Acquired KP1019 resistance was investigated by selecting KB-3-1 cells against KP1019 for more than 1 year. Only an approximately 2-fold KP1019 resistance could be induced, which unexpectedly was not due to overexpression of P-glycoprotein or other efflux pumps. Accordingly, KP1019-resistant cells did not display reduced drug accumulation. Their unique cross-resistance pattern confirmed an ABC transporter-independent resistance phenotype. In summary, the likeliness of acquiring insensitivity to KP1019 during therapy is expected to be low, and resistance should not be based on overexpression of drug efflux transporters.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4146
    Product Catalog Name:
    Anti-BCRP Antibody, clone BXP-21

  • Development of a rapid streptavidin capture-based assay for the tyrosine phosphorylated CSF-1R in peripheral blood mononuclear cells. 24339731

    A novel assay was developed to measure ratio of p-FMS (phospho FMS) to FMS using the Meso Scale Discovery(®) (MSD) technology and compared to the routinely used, IP-Western based approach. The existing IP-Western assay used lysed PBMCs (Peripheral Blood Mononuclear Cells) that were immunoprecipitated (IP) overnight, and assayed qualitatively by Western analysis. This procedure takes three days for completion. The novel IP-MSD method described in this paper employed immunoprecipitation of the samples for one hour, followed by assessment of the samples by a ruthenium labeled secondary antibody on a 96-well Streptavidin-coated MSD plate. This IP-MSD method was semi-quantitative, could be run in less than a day, required one-eighth the volume of sample, and compared well to the IP-Western method. In order to measure p-FMS/FMS, samples from healthy volunteers (HV) were first stimulated with CSF-1(Macrophage colony-stimulating factor) to initiate the changes in the phosphotyrosyl signaling complexes in FMS. The objective of the present work was to develop a high throughput assay that measured p-FMS/FMS semi-quantitatively, with minimal sample requirement, and most importantly compared well to the current IP-Western assay.
    Document Type:
    Reference
    Product Catalog Number:
    16-452
    Product Catalog Name:
    4G10® Platinum, Anti-Phosphotyrosine Antibody, Biotin Conjugate

  • CALHM1 controls the Ca²⁺-dependent MEK, ERK, RSK and MSK signaling cascade in neurons. 23345406

    Calcium homeostasis modulator 1 (CALHM1) is a Ca(2+) channel controlling neuronal excitability and potentially involved in the pathogenesis of Alzheimer's disease (AD). Although strong evidence indicates that CALHM1 is required for neuronal electrical activity, its role in intracellular Ca(2+) signaling remains unknown. In the present study, we show that in hippocampal HT-22 cells, CALHM1 expression led to a robust and relatively selective activation of the Ca(2+)-sensing kinases ERK1/2. CALHM1 also triggered activation of MEK1/2, the upstream ERK1/2-activating kinases, and of RSK1/2/3 and MSK1, two downstream effectors of ERK1/2 signaling. CALHM1-mediated activation of ERK1/2 signaling was controlled by the small GTPase Ras. Pharmacological inhibition of CALHM1 permeability using Ruthenium Red, Zn(2+), and Gd(3+), or expression of the CALHM1 N140A and W114A mutants, which are deficient in mediating Ca(2+) influx, prevented the effect of CALHM1 on the MEK, ERK, RSK and MSK signaling cascade, demonstrating that CALHM1 controlled this pathway via its channel properties. Importantly, expression of CALHM1 bearing the natural P86L polymorphism, which leads to a partial loss of CALHM1 function and is associated with an earlier age at onset in AD patients, showed reduced activation of ERK1/2, RSK1/2/3, and MSK1. In line with these results obtained in transfected cells, primary cerebral neurons isolated from Calhm1 knockout mice showed significant impairments in the activation of MEK, ERK, RSK and MSK signaling. The present study identifies a previously uncharacterized mechanism of control of Ca(2+)-dependent ERK1/2 signaling in neurons, and further establishes CALHM1 as a critical ion channel for neuronal signaling and function.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel. 2437119

    The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.
    Document Type:
    Reference
    Product Catalog Number:
    05-269

  • Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes. 20445169

    Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to less than or=100 pS when Cs(+) was replaced with Ca(2+). The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NP(o))-voltage curve was bell shaped, with NP(o) drastically decreasing when voltage was switched from -30 to -40 mV. NP(o) increased when intracellular calcium (Ca(2+)(i)) was raised within 0.1-100 microM to abruptly diminish with higher Ca(2+)(i). Thus maximal activity occurred within the Ca(2+)(i) range found in rat cerebral artery myocytes under physiological conditions. NP(o) was reduced by ruthenium red (80 muM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 microM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 muM), resistant to dantrolene (100 muM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • TRPV4 channels mediate the infrared laser-evoked response in sensory neurons. 22442563

    Infrared laser irradiation has been established as an appropriate stimulus for primary sensory neurons under conditions where sensory receptor cells are impaired or lost. Yet, development of clinical applications has been impeded by lack of information about the molecular mechanisms underlying the laser-induced neural response. Here, we directly address this question through pharmacological characterization of the biological response evoked by midinfrared irradiation of isolated retinal and vestibular ganglion cells from rodents. Whole cell patch-clamp recordings reveal that both voltage-gated calcium and sodium channels contribute to the laser-evoked neuronal voltage variations (LEVV). In addition, selective blockade of the LEVV by micromolar concentrations of ruthenium red and RN 1734 identifies thermosensitive transient receptor potential vanilloid channels as the primary effectors of the chain reaction triggered by midinfrared laser irradiation. These results have the potential to facilitate greatly the design of future prosthetic devices aimed at restoring neurosensory capacities in disabled patients.
    Document Type:
    Reference
    Product Catalog Number:
    AB5398P
    Product Catalog Name:
    Anti-Vanilloid Receptor Like Protein Antibody, pain, CT

  • Thermal sensitivity of isolated vagal pulmonary sensory neurons: role of transient receptor potential vanilloid receptors. 16513770

    A recent study has demonstrated that increasing the intrathoracic temperature from 36 degrees C to 41 degrees C induced a distinct stimulatory and sensitizing effect on vagal pulmonary C-fiber afferents in anesthetized rats (J Physiol 565: 295-308, 2005). We postulated that these responses are mediated through a direct activation of the temperature-sensitive transient receptor potential vanilloid (TRPV) receptors by hyperthermia. To test this hypothesis, we studied the effect of increasing temperature on pulmonary sensory neurons that were isolated from adult rat nodose/jugular ganglion and identified by retrograde labeling, using the whole cell perforated patch-clamping technique. Our results showed that increasing temperature from 23 degrees C (or 35 degrees C) to 41 degrees C in a ramp pattern evoked an inward current, which began to emerge after exceeding a threshold of approximately 34.4 degrees C and then increased sharply in amplitude as the temperature was further increased, reaching a peak current of 173 +/- 27 pA (n = 75) at 41 degrees C. The temperature coefficient, Q10, was 29.5 +/- 6.4 over the range of 35-41 degrees C. The peak inward current was only partially blocked by pretreatment with capsazepine (Delta I = 48.1 +/- 4.7%, n = 11) or AMG 9810 (Delta I = 59.2 +/- 7.8%, n = 8), selective antagonists of the TRPV1 channel, but almost completely abolished (Delta I = 96.3 +/- 2.3%) by ruthenium red, an effective blocker of TRPV1-4 channels. Furthermore, positive expressions of TRPV1-4 transcripts and proteins in these neurons were demonstrated by RT-PCR and immunohistochemistry experiments, respectively. On the basis of these results, we conclude that increasing temperature within the normal physiological range can exert a direct stimulatory effect on pulmonary sensory neurons, and this effect is mediated through the activation of TRPV1, as well as other subtypes of TRPV channels.
    Document Type:
    Reference
    Product Catalog Number:
    AB5398
    Product Catalog Name:
    Anti-Vanilloid Receptor Like Protein 1 Antibody, pain, CT

  • Spatio-temporal expression and functional involvement of transient receptor potential vanilloid 1 in diabetic mechanical allodynia in rats. 25020137

    Diabetic neuropathic pain (DNP) is one of the most common clinical manifestations of diabetes mellitus (DM), which is characterized by prominent mechanical allodynia (DMA). However, the molecular mechanism underlying it has not fully been elucidated. In this study, we examined the spatio-temporal expression of a major nociceptive channel protein transient receptor potential vanilloid 1 (TRPV1) and analyzed its functional involvement by intrathecal (i.t.) application of TRPV1 antagonists in streptozocin (STZ)-induced DMA rat models. Western blot and immunofluorescent staining results showed that TRPV1 protein level was significantly increased in the soma of the dorsal root ganglion (DRG) neurons on 14 days after STZ treatment (DMA 14 d), whereas those in spinal cord and skin (mainly from the central and peripheral processes of DRG neurons) had already been enhanced on DMA 7 d to peak on DMA 14 d. qRT-PCR experiments confirmed that TRPV1 mRNA level was significantly up-regulated in the DRG on DMA 7 d, indicating a preceding translation of TRPV1 protein in the soma but preferential distribution of this protein to the processes under the DMA conditions. Cell counting assay based on double immunostaining suggested that increased TRPV1-immunoreactive neurons were likely to be small-sized and CGRP-ergic. Finally, single or multiple intrathecal applications of non-specific or specific TRPV1 antagonists, ruthenium red and capsazepine, at varying doses, effectively alleviated DMA, although the effect of the former was more prominent and long-lasting. These results collectively indicate that TRPV1 expression dynamically changes during the development of DMA and this protein may play important roles in mechanical nociception in DRG neurons, presumably through facilitating the release of CGRP.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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