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  • Immunochemical mapping of domains in human interleukin 4 recognized by neutralizing monoclonal antibodies. 7682108

    Human interleukin 4 is a highly pleiotropic cytokine secreted by activated T cells that exerts multiple biological effects on B and T lymphocytes and other cell types. Elucidation of structure-function relations was accomplished by epitope mapping of a panel of monoclonal antibodies and by mutagenesis of selected amino acid residues. Epitope mapping of these monoclonal antibodies was achieved through binding studies with recombinant human interleukin 4 (rhuIL-4), proteolytic fragments produced by digestion with Staphylococcus aureus V8 protease and synthetic peptides derived from the sequence of the parent molecule. Monoclonal antibodies 25D2, 35F2, and 11B4 neutralized the in vitro T-cell proliferation activity of rhuIL-4 and also prevented binding of rhuIL4 to its cell surface receptor. These antibodies recognized sequences 104-129, 70-92, and 61-82, respectively. These regions comprise the BC loop/helix C (residues 61-92) and helix D (residues 104-129). A nonneutralizing monoclonal antibody (1A2) recognized a nonoverlapping region (residues 43-59) comprising almost entirely helix B. Mutagenesis of a cluster of residues within helix C showed that at least three residues (K84, R88, and N89) were potentially involved in receptor recognition. The existence of two distinct nonneighboring binding domains in the three-dimensional structure of rhuIL-4 provided preliminary evidence for a model of receptor interaction involving the formation of a ternary complex consisting of two molecules of the extracellular portion of the receptor and one molecule of rhuIL-4.
    Document Type:
    Reference
    Product Catalog Number:
    20-103
    Product Catalog Name:
    Phosphate Standard

  • Urinary metanephrine and normetanephrine determined without extraction by using liquid chromatography and coulometric array detection. 8375055

    We describe a procedure for the direct measurement of metanephrine (MN) and normetanephrine (NMN) in hydrolyzed urine, using HPLC with coulometric array detection. Acid-hydrolyzed samples were diluted and filtered before separation by isocratic reversed-phase ion-pair chromatography. Eight serial coulometric sensors, set at incrementally increasing anodic potentials, were used to screen lower-oxidizing interferences and provide stepwise oxidation of the metanephrines. Voltammetric behavior across three adjacent sensors was used to assess resolution and aid in peak identification. Values obtained in commercial controls were consistently within the specified target range. Variability, expressed as CV, was 5.45-9.22% between runs and 1.60-4.52% within-run for both compounds. The limit of detection was 2.6 micrograms/L for MN and 2.8 micrograms/L for NMN, with a linear response to 15.0 mg/L for both analytes. Results from patients' samples correlated well with those by a method involving dual ion-exchange extraction (r = 0.963, n = 82 for MN; r = 0.9768, n = 83 for NMN). This procedure provided high selectivity and objective peak purity information while greatly simplifying sample preparation.
    Document Type:
    Reference
    Product Catalog Number:
    20-176
    Product Catalog Name:
    100X GTPγS, 10mM

  • Genomic structure and chromosomal mapping of the human CD22 gene. 8496602

    The human CD22 gene is expressed specifically in B lymphocytes and likely has an important function in cell-cell interactions. A nearly full length human CD22 cDNA clone was used to isolate genomic clones that span the CD22 gene. The CD22 gene is spread over 22 kb of DNA and is composed of 15 exons. The first exon contains the major transcriptional start sites. The translation initiation codon is located in exon 3, which also encodes a portion of the signal peptide. Exons 4 to 10 encode the seven Ig domains of CD22, exon 11 encodes the transmembrane domain, exons 12 to 15 encode the intracytoplasmic domain of CD22, and exon 15 also contains the 3' untranslated region. A minor form of CD22 mRNA likely results from splicing of exon 5 to exon 8, skipping exons 6 and 7. A 4.6-kb XbaI fragment of the CD22 gene was used to map the chromosomal location of CD22 by fluorescence in situ hybridization. The hybridization locus was identified by combining fluorescent images of the probe with the chromosomal banding pattern generated by an Alu probe. The results demonstrate that CD22 is located within the band region q13.1 of chromosome 19. Two closely clustered major transcription start sites and several minor start sites were mapped by primer extension. Similarly to many other lymphoid-specific genes, the CD22 promoter lacks an obvious TATA box. Approximately 4 kb of DNA 5' of the transcription start sites were sequenced and found to contain multiple Alu elements. Potential binding sites for the transcriptional factors NF-kappa B, AP-1, and Oct-2 are located within 300 bp 5' of the major transcription start sites. A 400-bp fragment (bp -339 through +71) of the CD22 promoter region was subcloned into a pGEM-chloramphenicol acetyltransferase vector and after transfection into B and T cells was found to be active in both B and T cells. Further studies of the CD22 gene should lead to a greater understanding of the expression of CD22 during B cell development and differentiation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Casein kinase II beta-subunit inhibits the activity of the catalytic alpha-subunit in the absence of salt. 8268212

    Casein kinase II (CKII) has a subunit structure of alpha 2 beta 2 where alpha is the catalytic subunit. Recombinant Drosophila casein kinase II alpha-subunit expressed in insect cells is inhibited by NaCl, thermally labile and inactivated by binding to plastic. In the presence of detergent (Tween 80) recombinant alpha-subunit has a kcat of 249 min-1 (Km 170 microM) for the peptide substrate RRRDDDSDDD-NH2, compared to recombinant Drosophila CKII with a kcat of 71 min-1 (per mol alpha) (Km 42 microM) and bovine CKII with a kcat of 123 min-1 (per mol alpha) (Km 45 microM) when measured in the absence of NaCl. The kcat values of bovine CKII and recombinant Drosophila CKII measured in the presence of 150 mM NaCl were 429 min-1 (per mol alpha) (Km 82 microM) and 204 min-1 (per mol alpha) (Km 51 microM), respectively. Since the kcat for the Drosophila alpha-subunit is approx. 3-fold greater than the Drosophila CKII measured in the absence of added salt these results indicate that the beta-subunit acts primarily as an inhibitory subunit.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Measurement of insulin-like growth factor-II in human plasma using a specific monoclonal antibody-based two-site immunoradiometric assay. 8492071

    An immunoradiometric assay (IRMA) for the measurement of insulin-like growth factor-II (IGF-II) in human plasma has been developed, optimized and evaluated clinically in normal subjects and patients with disorders of the GH/IGF-I axis. Six monoclonal antibodies (MAbs) to recombinant human IGF-II (rhIGF-II) were produced, all of which had low cross-reactivity with rhIGF-I (< 0.01%) and insulin (< 0.01%). Compatibility of pairs of MAbs was tested in two-site IRMAs using three radioiodinated MAbs and three MAbs linked to Sephacryl S-300 (with separation of bound and free radiolabelled MAb by sucrose layering). Seven pairs of MAbs bound rhIGF-II and the combination of 125I-labelled W3D9 and W2H1 linked to solid phase was selected. The optimized assay had a completion time of 4 h, a minimum detection limit of 30 ng/ml (2.5 standard deviations from the zero standard) and detected a single peak of endogenous IGF-II in normal plasma which co-eluted with rhIGF-II after acid gel chromatography. IGF-II was measured in formic acid/acetone extracts of plasma from 16 normal subjects (mean 685, range 516-1008 micrograms/l), four acromegalic patients (mean 637, range 553-700 micrograms/l), fourteen patients with type-1 diabetes (mean 635, range 247-753 micrograms/l), nine patients with uraemia (mean 423, range 78-850 micrograms/l), and three patients with Laron-type GH insensitivity (75, 35 and 36 micrograms/l). No significant fluctuations were detected between samples obtained hourly from 08.00 to 19.00 h in normal subjects. Low levels of IGF-binding proteins (IGFBPs) remaining in plasma extracts may interfere with the measurement of IGF-II and give rise to falsely elevated IGF-II levels in radioimmunoassays or falsely suppressed levels in IRMAs. Such interference did not occur with the IRMA when used to measure IGF-II in extracts from normal subjects, acromegalic patients and patients with type-1 diabetes, and the addition of excess rhIGF-I in order to displace IGF-II from residual IGFBPs had no effect on IGF-II measurements in these samples. However, levels of IGF-II measured in extracts from patients with Laron-type GH insensitivity and patients with uraemia increased markedly after preincubation with excess rhIGF-I. The accurate measurement of IGF-II by IRMA in extracts from these subjects therefore requires the displacement of IGF-II from IGFBPs prior to assay. We conclude that, in contrast to radioimmunoassays, the two-site IRMA developed here provides a practical, rapid and specific method for the measurement of IGF-II in human plasma.
    Document Type:
    Reference
    Product Catalog Number:
    CBL82

  • MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture. 8499602

    Cultures of hippocampal neurones have been used to study a possible correlation between the pattern of dendritic growth and post-translational modification of Microtubule-Associated Protein 2 (MAP2). During the first three days in vitro, a small increase in the total amount of MAP2 is observed, whereas the level of phosphorylation increases exponentially, being particularly dramatic after three days in vitro. Analysis of dendrite morphology by MAP2 immunofluorescence, revealed a parallel exponential growth in dendrite arborization with maximum rates at the third day of culture. We propose a correlation between dendrite arborization and phosphorylation of MAP2, that could be mediated by the establishment of cell contacts.
    Document Type:
    Reference
    Product Catalog Number:
    AB5622
    Product Catalog Name:
    Anti-Microtubule-Associated Protein 2 (MAP2) Antibody

  • LAZ3, a novel zinc-finger encoding gene, is disrupted by recurring chromosome 3q27 translocations in human lymphomas. 8220427

    We have shown previously that chromosomal translocations involving chromosome 3q27 and immunoglobulin gene regions are the third most common specific translocations in non-Hodgkin's lymphoma (NHL). We now report the isolation of a gene that is disrupted in two cases by t(3;14) and t(3;4) translocations. The gene (LAZ3) encodes a 79 kDa protein containing six zinc-finger motifs and sharing amino-terminal homology with several transcription factors including the Drosophila tramtrack and Broad-complex genes, both of which are developmental transcription regulators. LAZ3 is transcribed as a 3.8 kb message predominantly in normal adult skeletal muscle and in several NHL carrying 3q27 chromosomal defects. We suggest that it may act as a transcription regulator and play an important role in lymphomagenesis.
    Document Type:
    Reference
    Product Catalog Number:
    04-437

  • Co-clustering of beta 1 integrins, cytoskeletal proteins, and tyrosine-phosphorylated substrates during integrin-mediated leukocyte aggregation. 7690816

    The involvement of the VLA-4 integrin in an alternative leukocyte homotypic adhesion mechanism that is LFA-1/ICAM-1 independent, has been previously reported. We describe here the localization of beta 1 and alpha 4 integrin subunits at sites of cell-cell contact, on both beta 1- and alpha 4-induced aggregates of B lymphoblastoid Ramos cells. Moreover, the distribution of different VLA-alpha subunits was also examined on beta 1-induced cell aggregates of alpha 2- and alpha 4-transfected K-562 cells. Both alpha 2 and alpha 4 integrin subunits were mainly localized at sites of intercellular boundaries, suggesting a possible role for these integrins in leukocyte intercellular adhesion. The fibronectin receptor alpha 5 subunit was either diffuse throughout the plasma membrane, or displayed some accumulation at sites of cell-cell contact. Even though homotypic aggregation of U-937 cells was induced with the anti-alpha 5 P1D6 mAb, the alpha 5 subunit showed only partial redistribution to regions of cell-cell contact, compared with the complete redistribution of the alpha 4 subunit in the alpha 4-induced aggregates. The reorganization of the actin-cytoskeleton was observed at sites of intercellular boundaries in both the anti-beta 1- and anti-alpha 4-induced cell aggregates. Hence, F-actin and the cytoskeletal protein talin co-localized with beta 1 and alpha 4 integrin clusters at sites of cell-cell contact. Signal transduction during VLA-mediated homotypic cell adhesion has also been investigated. We found co-localization of beta 1 and alpha 4 subunits with tyrosine-phosphorylated proteins at cell-cell contact regions during cell aggregation. These data indicate that VLA integrin-mediated leukocyte aggregation results in clustering of beta 1-integrins at sites of cell-cell contact, together with co-localization of cytoskeletal proteins. These results also suggest that protein tyrosine phosphorylation is an important signal transduction mechanism when VLA integrins participate in intercellular contacts.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Organization of the murine Cd22 locus. Mapping to chromosome 7 and characterization of two alleles. 8100843

    Murine CD22 (mCD22) is a B cell-associated adhesion protein with seven extracellular Ig-like domains that has 62% amino acid identity to its human homologue. Southern analysis on genomic DNA isolated from tissues and cell lines from several mouse strains using mCD22 cDNA demonstrated that the Cd22 locus encoding mCD22 is a single copy gene of < or = 30 kb. Digestion of genomic DNA preparations with four restriction endonucleases revealed the presence of restriction fragment length polymorphisms (RFLP) in BALB/c, C57BL/6, and C3H strains vs DBA/2J, NZB, and NZC strains, suggesting the presence of two or more Cd22 alleles. Using a mCD22 cDNA clone derived from the BALB/c strain, we isolated genomic clones from a DBA/2J genomic library that contained all the exons necessary to encode the full length mCD22 cDNA. Fifteen exons, including exon 3 that encodes the translation start codon, were identified. Each extracellular Ig-like domain of mCD22 is encoded by a single exon. A comparison between the nucleotide sequences of the BALB/c CD22 cDNA and the exons of the DBA/2J CD22 genomic clones revealed an 18-nucleotide deletion in exon 4 (encoding the most distal Ig-like domain 1 of mCD22) of the DBA/2J genomic sequence in addition to a number of substitutions, insertions, and deletions in other exons. These nucleotide differences were also present in a cDNA clone isolated from total RNA of LPS-activated DBA/2J splenocytes by reverse transcription-polymerase chain reaction. The Cd22 locus was mapped to the proximal region of chromosome 7, a region sytenic to human chromosome 19q, close to the previously reported loci, Lyb-8 and Mag (a homologue of Cd22). An antibody (CY34) against the Lyb-8.2 B cell marker reacted with a BHK transfectant expressing the full length mCD22 cDNA, thus demonstrating that Lyb-8 and Cd22 loci are identical. Furthermore, a rat anti-mCD22 mAb, NIM-R6, bound to sIgM+ DBA/2J B cells, confirming the expression of a CD22 protein by the Cd22a/Lyb-8a allele.
    Document Type:
    Reference
    Product Catalog Number:
    MABF980

  • Interactions of nutrients, insulin-like growth factors (IGFs) and IGF-binding proteins in the regulation of DNA synthesis by isolated fetal rat islets of Langerhans. 7506287

    Insulin is a major regulatory hormone for optimal tissue growth and function in utero. Its continued availability to the growing fetus depends on increasing islet cell mass. The purpose of the study was to examine the interactions between nutrient availability and insulin-like growth factor (IGF) release and action during DNA synthesis by isolated fetal rat islets of Langerhans. Specifically, we wished to determine (a) whether the availability of glucose or total amino acids altered the release of endogenous IGF-I or -II, (b) if both IGF-I and -II were effective mitogens for pancreatic beta-cells, (c) whether islets released IGF-binding proteins (IGFBPs) and their possible regulation by nutrient availability and (d) how IGFBPs might regulate the ability of IGFs to alter islet DNA synthesis. Islets of Langerhans were isolated from fetal rat pancreata on day 22 of gestation by collagenase digestion. Islets enriched in beta-cells following a 5-day preincubation regime were maintained in various concentrations of glucose (1.4-16.7 mmol/l) or amino acids (x1- x3 total concentrations), with or without exogenous IGF-I, -II, IGFBP-1 or IGFBP-2. The release of insulin and endogenous IGF-I and -II were each determined by radioimmunoassay, and IGFBP release characterized by Western ligand blot analysis. DNA synthesis was measured by the incorporation of [3H]thymidine. Isolated islets demonstrated an increased release of insulin in response to increasing amounts of both glucose and amino acids, demonstrating functional viability. Both classes of nutrients also increased the DNA synthetic rate of islets. Islets released almost twice as much IGF-II (0.22 +/- 0.08 nmol/l, mean +/- S.E.M., n = 4) as IGF-I (0.14 +/- 0.03 nmol/l) in cultures containing 8.7 mmol glucose/l and x1 amino acids. Lesser or greater concentrations of glucose did not alter the release of either IGF, but the release of IGF-II was significantly increased (0.53 +/- 0.08 nmol/l, P < 0.01) in the presence of x2 amino acids. Exogenous IGF-I was fivefold more active in stimulating DNA synthesis by islets (half maximal concentration (ED50) 1.6 +/- 0.4 nmol/l, n = 3) than was IGF-II (ED50 8.1 +/- 0.6 nmol/l), regardless of glucose concentration. Isolated islets released four species of IGFBP with molecular sizes of approximately 19, 25, 35 and 46 kDa respectively. The 35 kDa form was identified by Western immunoblot as IGFBP-2. Increasing the glucose concentration between 1.4 mmol/l and 16.7 mmol/l caused a dose-related increase in the release of the 19, 25 and 35 kDa IGFBP species.(ABSTRACT TRUNCATED AT 400 WORDS)
    Document Type:
    Reference
    Product Catalog Number:
    06-108