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  • TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors. 2580933

    The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.
    Document Type:
    Reference
    Product Catalog Number:
    MABT398
    Product Catalog Name:
    Anti-CD226/DNAM-1 Antibody, clone LeoA1 (Azide Free)
  • The human cardiac and skeletal muscle proteomes defined by transcriptomics and antibody-based profiling. 26109061

    To understand cardiac and skeletal muscle function, it is important to define and explore their molecular constituents and also to identify similarities and differences in the gene expression in these two different striated muscle tissues. Here, we have investigated the genes and proteins with elevated expression in cardiac and skeletal muscle in relation to all other major human tissues and organs using a global transcriptomics analysis complemented with antibody-based profiling to localize the corresponding proteins on a single cell level.Our study identified a comprehensive list of genes expressed in cardiac and skeletal muscle. The genes with elevated expression were further stratified according to their global expression pattern across the human body as well as their precise localization in the muscle tissues. The functions of the proteins encoded by the elevated genes are well in line with the physiological functions of cardiac and skeletal muscle, such as contraction, ion transport, regulation of membrane potential and actomyosin structure organization. A large fraction of the transcripts in both cardiac and skeletal muscle correspond to mitochondrial proteins involved in energy metabolism, which demonstrates the extreme specialization of these muscle tissues to provide energy for contraction.Our results provide a comprehensive list of genes and proteins elevated in striated muscles. A number of proteins not previously characterized in cardiac and skeletal muscle were identified and localized to specific cellular subcompartments. These proteins represent an interesting starting point for further functional analysis of their role in muscle biology and disease.
    Document Type:
    Reference
    Product Catalog Number:
    07-087
  • The human T-cell leukemia virus-1 transcriptional activator Tax enhances cAMP-responsive element-binding protein (CREB) binding activity through interactions with the DNA ... 9668114

    Tax-1, the transcriptional activation protein of human T-cell leukemia virus-1, increases transcription from the human T-cell leukemia virus-1 long terminal repeat and specific cellular promoters through interactions with cellular DNA-binding proteins. The Tax response elements (TxREs) of the long terminal repeat resemble cAMP response elements (CREs), the target of cAMP-responsive element-binding protein (CREB). CREB binds the TxRE with reduced affinity; however, the interaction is specifically enhanced by Tax. Using a fluorescence quenching method, we determined that CREB dimerizes in the absence of DNA, and that Tax does not enhance dimerization. DNA footprinting of the TxRE with 1, 10-phenanthroline-copper complex demonstrates that Tax contacts DNA and extends the footprint of CREB to GC-rich sequences flanking the core CRE-like element. The minor groove-binding drug chromomycin A3, but not distamycin A, disrupted Tax-enhanced CREB binding to the TxRE. Substitution of the guanine-rich sequences flanking the core of the TxRE with inosine residues also blocked the Tax effect. Finally, the IC-substituted TxRE binds CREB with increased affinity, suggesting flanking DNA influences the binding of CREB to the core CRE-like element. These data indicate that Tax does not regulate DNA binding of CREB by altering dimerization, but rather enhances DNA binding by additionally interacting with the minor groove of flanking DNA sequences.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • The Human Accelerated Region 1 noncoding RNA is repressed by REST in Huntington's disease. 20179156

    In the neurons of Huntington's disease (HD) patients, gene regulatory networks are disrupted by aberrant nuclear localization of the master transcriptional repressor, REST. Emerging evidence suggests that, in addition to protein-coding genes, non-coding RNAs (ncRNAs) may also contribute to neurodegenerative processes. To discover ncRNAs that are involved in HD, we screened genome-wide data for novel, non-coding targets of REST. This identified Human Accelerated Region 1 (HAR1), a rapidly-evolving cis-antisense locus that is specifically transcribed in the nervous system. We show that REST is targeted to the HAR1 locus by specific DNA regulatory motifs, resulting in potent transcriptional repression. Consistent with other REST target genes, HAR1 levels are significantly lower in the striatum of HD patients compared to unaffected individuals. These data represent further evidence that non-coding gene expression changes accompany neurodegeneration in Huntington's disease. Key words: Huntington's Disease, HAR1, noncoding RNA.
    Document Type:
    Reference
    Product Catalog Number:
    07-579
    Product Catalog Name:
    Anti-REST Antibody
  • A human artificial chromosome recapitulates the metabolism of native telomeres in mammalian cells. 24558398

    Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
  • The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. 9230079

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.
    Document Type:
    Reference
    Product Catalog Number:
    07-272