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  • The VPAC2 agonist peptide histidine isoleucine (PHI) up-regulates glutamate transport in the corpus callosum of a rat model of amyotrophic lateral sclerosis (hSOD1G93A) b ... 21730107

    Degeneration of corpus callosum appears in patients with amyotrophic lateral sclerosis (ALS) before clinical signs of upper motor neuron death. Considering the ALS-associated impairment of astrocytic glutamate uptake, we have characterized the expression and activity of the glutamate transporter isoforms GLT-1a and GLT-1b in the corpus callosum of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1(G93A)). We have also studied the effect of peptide histidine isoleucine (PHI), a vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 2 (VPAC(2)) agonist on glutamate transporters both in vivo and in callosal astrocytes. Before the onset of motor symptoms, the expression of both transporter isoforms was correlated with a constitutive activity of caspase-3. This enzyme participates in the down-regulation of GLT-1 in ALS, and here we demonstrated its involvement in the selective degradation of GLT-1a in the white matter. A single stereotactic injection of PHI into the corpus callosum of symptomatic rats decreased caspase-3 activity and promoted GLT-1a expression and uptake activity. Together, with evidence for a reduced expression of prepro-VIP/PHI mRNA in the corpus callosum of transgenic animals, these data shed light on the modulatory role of the VIP/PHI system on the glutamatergic transmission in ALS.
    Document Type:
    Reference
    Product Catalog Number:
    AB1783
    Product Catalog Name:
    Anti-Glutamate Transporter Antibody, Glial

  • Phosphorylation-dependent regulation of ryanodine receptors: a novel role for leucine/isoleucine zippers. 11352932

    Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.
    Document Type:
    Reference
    Product Catalog Number:
    06-852
    Product Catalog Name:
    Anti-Spinophilin/Neurabin-II Antibody

  • BCAA catabolism in brown fat controls energy homeostasis through SLC25A44 31435015

    Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism. 22654736

    In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5406
    Product Catalog Name:
    Anti-GAD67 Antibody, clone 1G10.2

  • RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody. 11027361

    Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.
    Document Type:
    Reference
    Product Catalog Number:
    AB4073
    Product Catalog Name:
    Anti-Farnesyl Antibody

  • The cell attachment domain of fibronectin. Determination of the primary structure. 7050098

    The complete amino acid sequence of the cell attachment domain of human plasma fibronectin (Pierschbacher, M. D., Hayman, E. G., and Ruoslahti, E. (1981) Cell 26, 259-267) has been determined by automated sequential degradation of a peptic fragment comprising this region and of peptides derived from this fragment by digestion with thermolysin, staphylococcal V8 protease, cyanogen bromide cleavage, and partial acid hydrolysis. The fragment contains 108 residues with isoleucine and methionine as the NH2- and carboxyl-terminal amino acids, respectively. No cysteines are present. The calculated molecular weight of the cell attachment fragment, based on the amino acid sequence, is 11,482, which is in good agreement with the molecular weight estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and ultracentrifugation. There are no homologies in this fragment with other published sequences. The implications of the structure of the cell attachment fragment to the molecular mechanism of cell-fibronectin interaction are discussed.
    Document Type:
    Reference
    Product Catalog Number:
    MAB88916-C
    Product Catalog Name:
    Anti-Fibronectin (Cell Attachment Fragment) Antibody, clone 3E3, Ascites & Azide Free

  • Dynamic interaction of the measles virus hemagglutinin with its receptor signaling lymphocytic activation molecule (SLAM, CD150). 18292085

    The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8905
    Product Catalog Name:
    Anti-Measles Blend Antibody, hemagglutinin, clones CV1, CV4

  • A mouse model of the human Fragile X syndrome I304N mutation. 20011099

    The mental retardation, autistic features, and behavioral abnormalities characteristic of the Fragile X mental retardation syndrome result from the loss of function of the RNA-binding protein FMRP. The disease is usually caused by a triplet repeat expansion in the 5'UTR of the FMR1 gene. This leads to loss of function through transcriptional gene silencing, pointing to a key function for FMRP, but precluding genetic identification of critical activities within the protein. Moreover, antisense transcripts (FMR4, ASFMR1) in the same locus have been reported to be silenced by the repeat expansion. Missense mutations offer one means of confirming a central role for FMRP in the disease, but to date, only a single such patient has been described. This patient harbors an isoleucine to asparagine mutation (I304N) in the second FMRP KH-type RNA-binding domain, however, this single case report was complicated because the patient harbored a superimposed familial liver disease. To address these issues, we have generated a new Fragile X Syndrome mouse model in which the endogenous Fmr1 gene harbors the I304N mutation. These mice phenocopy the symptoms of Fragile X Syndrome in the existing Fmr1-null mouse, as assessed by testicular size, behavioral phenotyping, and electrophysiological assays of synaptic plasticity. I304N FMRP retains some functions, but has specifically lost RNA binding and polyribosome association; moreover, levels of the mutant protein are markedly reduced in the brain specifically at a time when synapses are forming postnatally. These data suggest that loss of FMRP function, particularly in KH2-mediated RNA binding and in synaptic plasticity, play critical roles in pathogenesis of the Fragile X Syndrome and establish a new model for studying the disorder.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2160
    Product Catalog Name:
    Anti-Fragile X Mental Retardation Protein Antibody, clone 1C3

  • Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate. 21072200

    Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.
    Document Type:
    Reference
    Product Catalog Number:
    AB1504
    Product Catalog Name:
    Anti-Glutamate receptor 1 Antibody

  • Molecular Characteristics of Clostridium perfringens TpeL Toxin and Consequences of Mono-O-GlcNAcylation of Ras in Living Cells. 22665487

    TpeL is a member of the family of clostridial glucosylating toxins produced by Clostridium perfringens type A, B, and C strains. In contrast to other members of this toxin family, it lacks a C-terminal polypeptide repeat domain, which is suggested to be involved in target cell binding. It was shown that the glucosyltransferase domain of TpeL modifies Ras in vitro by mono-O-glucosylation or mono-O-GlcNAcylation (Nagahama, M., Ohkubo, A., Oda, M., Kobayashi, K., Amimoto, K., Miyamoto, K., and Sakurai, J. (2011) Infect. Immun. 79, 905-910). Here we show that TpeL preferably utilizes UDP-N-acetylglucosamine (UDP-GlcNAc) as a sugar donor. Change of alanine 383 of TpeL to isoleucine turns the sugar donor preference from UDP-GlcNAc to UDP-glucose. In contrast to previous studies, we show that Rac is a poor substrate in vitro and in vivo and requires 1-2 magnitudes higher toxin concentrations for modification by TpeL. The toxin is autoproteolytically processed in the presence of inositol hexakisphosphate (InsP(6)) by an intrinsic cysteine protease domain, located next to the glucosyltransferase domain. A C-terminally extended TpeL full-length variant (TpeL1-1779) induces apoptosis in HeLa cells (most likely by mono-O-GlcNAcylation of Ras), and inhibits Ras signaling including Ras-Raf interaction and ERK activation. In addition, TpeL blocks Ras signaling in rat pheochromocytoma PC12 cells. TpeL is a glucosylating toxin, which modifies Ras and induces apoptosis in target cells without having a typical C-terminal polypeptide repeat domain.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5