L T Kakach

University of Minnesota Duluth, Duluth, Minnesota, United States

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Publications (8)22.67 Total impact

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    ABSTRACT: Tumor necrosis factor-like ligand 1A (TL1A) is a type II transmembrane protein predominantly expressed by endothelial cells that promotes the expansion of activated T cells and regulatory T cells, modulates inflammation, and regulates the production of a wide variety of T cell cytokines. However, there have not been any mAbs which specifically detect chTL1A and define its biochemical and immunological properties. So in this study, two mouse monoclonal antibodies (mAbs) which specifically detect chicken TL1A (chTL1A) were developed and characterized. Both mAbs identified a 32 kDa E. coli-derived, poly-histidine-tagged fusion protein by Western blot analysis. The mAbs identified TL1A-secreting cells in the chicken thymus, cecal tonsil, and bursa of Fabricius by immunocytochemistry, and were used to measure serum TL1A levels in normal and necrotic enteritis (NE)-afflicted chickens by antigen capture ELISA. These mAbs inhibited chTL1A-induced spleen lymphocyte proliferation, nitric oxide production by chicken macrophage cells (HD11), and blocked the cytotoxic effect of chTL1A against lymphoblastoid chicken B tumor cells (LSCC-RP9). These new mAbs that detect chTL1A will be important immune reagents for basic and applied research in poultry immunology.
    Veterinary Immunology and Immunopathology 01/2014; · 1.88 Impact Factor
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    ABSTRACT: This report describes the cloning and characterization of expressed gene sequences of the swine and bovine interferon-gamma inducible chemokine CXCL11, or I-TAC, associated with type 1 T-helper immune responses, and affirmation of bioactivity of their yeast-expressed protein products. The coding regions of both cDNA sequences were 303 nucleotides in length; each is coded for four exons in the genome. The bovine coding region shared 82% and 70% homology with human and mouse CXCL11, respectively, and the swine coding region 84% and 72% homology, respectively. As expected the swine and bovine CXCL11 sequences showed less homology with other human and mouse C-X-C motif chemokine sequences. Each cDNA was cloned into plasmids and transfected into Pichia pastoris (yeast) and the resultant expressed protein purified. Biological activity of each purified chemokine was affirmed by chemotaxis assays. Both swine and bovine CXCL11 were chemotactic for mitogen and IL-2 stimulated peripheral blood mononuclear cells. This is the first report for bioactivity of this chemokine in livestock species. This work provides valuable new reagents for investigating basic immunity as well as vaccine and disease responses in swine and cattle, goals of the U.S. Veterinary Immune Reagent Network which supported this effort.
    Veterinary Immunology and Immunopathology 03/2010; 136(1-2):170-5. · 1.88 Impact Factor
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    M P Murtaugh, M R Elam, L T Kakach
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    ABSTRACT: The 3'-portion of the genome of a U.S. isolate of the porcine reproductive and respiratory syndrome (PRRS) virus, ATCCVR-2332, was cloned and sequenced. The resultant 3358 nucleotides contain 6 open reading frames (ORFs) with homologies to ORFs 2 through 7 of the European strain of the PRRS virus and other members of the free-standing genus of arteriviruses. Both VR-2332 and the European isolate (called the Lelystad virus) have been identified as infectious agents responsible for the swine disease called PRRS. Comparative sequence analysis indicates that there are degrees of amino acid identity to the Lelystad virus open reading frames ranging from 55% in ORF 5 to 79% in ORF 6. Hydropathy profiles indicate that the ORFs of VR-2332 and Lelystad virus correspond structurally despite significant sequence differences. These results are consistent with the biological similarities but distinct serological properties of North American and European isolates of the virus.
    Archives of Virology 02/1995; 140(8):1451-60. · 2.03 Impact Factor
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    J A Suzich, L T Kakach, M S Collett
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    ABSTRACT: The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.
    Journal of Virology 05/1990; 64(4):1549-55. · 5.08 Impact Factor
  • L T Kakach, J A Suzich, M S Collett
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    ABSTRACT: The M segment RNA of Rift Valley fever virus (RVFV) encodes four gene products: the two viral envelop glycoproteins G2 and G1, a glycosylated 78-kDa protein, and a nonglycosylated 14-kDa protein. These proteins are generated from a single open reading frame (ORF) by a strategy involving independent translational initiations at both the first and second in-phase ATG codons and co-translational processing of primary polyprotein products. The ORF encodes six sites for N-linked glycosylation: one present in the "preglycoprotein region" preceding the coding sequences of the mature envelop glycoproteins, and within the coding sequences of both the 78- and 14-kDa proteins; one site in the glycoprotein G2 coding region, also present in the 78-kDa protein; and four sites within glycoprotein G1. From analyses of RVFV proteins produced in cells infected with recombinant vaccinia viruses expressing various M segment regions, we show glycoprotein G2 was glycosylated at its single site and glycoprotein G1 at at least three sites. Both sites for N-linked glycosylation in the 78-kDa protein were occupied with glycan. This latter result indicated the preglycoprotein region glycosylation site was utilized in the 78-kDa protein, but this same site within the 14-kDa protein was not. Further analysis showed utilization of this glycosylation site, as well as proteolytic processing at the amino terminus of the mature glycoprotein G2, appeared to be determined by initiation codon usage. The two-site translational initiation expression strategy of this phlebovirus M segment and its role in the control of post-translational protein modification and processing are discussed.
    Virology 07/1989; 170(2):505-10. · 3.37 Impact Factor
  • T L Wasmoen, L T Kakach, M S Collett
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    ABSTRACT: The Phlebovirus Rift Valley fever virus (RVFV), like other members of the Bunyaviridae family, matures intracellularly at the smooth-surfaced vesicles in the Golgi region of infected cells. Here we show that in cultured cells the RVFV glycoproteins G2 and G1 accumulate and are retained at this site. To investigate the parameters governing this subcellular localization, we have engineered portions of the cloned RVFV M segment (which encodes a 14- and a 78-kDa protein, in addition to glycoproteins G2 and G1) into vaccinia virus. Immunofluorescent analysis of cells infected with a vaccinia virus recombinant containing the entire open reading frame of the RVFV M segment revealed Golgi localization for glycoproteins G2, G1, the 78-kDa protein, and Golgi as well as some reticular distribution for the 14-kDa protein. These distributions paralleled those seen in authentic RVFV-infected cells. RVFV-vaccinia virus recombinants possessing progressive deletions within the 152 amino acid preglycoprotein sequence of the M segment were analyzed for possible effects on the cellular distribution of G2 and G1. Removal of the first 130 amino acids of the open reading frame amino-terminal to the mature glycoprotein coding sequences, while abolishing production of the 78- and 14-kDa proteins, did not alter the Golgi location of G2 and G1. The data suggest that Golgi-specific signals reside within the G2 and/or G1 glycoprotein sequences. The use of vaccinia virus recombinants provides a genetically manipulable expression system with which to further investigate the sequences involved in the intracellular localization of these Phlebovirus proteins.
    Virology 10/1988; 166(1):275-80. · 3.37 Impact Factor
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    L T Kakach, T L Wasmoen, M S Collett
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    ABSTRACT: Recombinant vaccinia viruses were constructed and used in conjunction with site-specific antisera to study the coding capacity and detailed expression strategy of the M segment of the Phlebovirus Rift Valley fever virus (RVFV). The M segment could be completely and faithfully expressed in recombinant RVFV-vaccinia virus-infected cells, the gene products apparently being correctly processed and modified in the absence of the RVFV L and S genomic segments. The proteins encoded by the RVFV M segment included, in addition to the viral glycoproteins G2 and G1, two previously uncharacterized polypeptides of 78 and 14 kilodaltons (kDa). By manipulation of RVFV sequences present in the recombinant vaccinia viruses and use of specific antibody reagents, it was found that the 78-kDa protein initiated at the first initiation codon of the open reading frame and encompassed the entire preglycoprotein and glycoprotein G2 coding sequences. The 14-kDa protein appeared to begin from the second in-phase ATG and was composed of only the preglycoprotein sequences. Both viral glycoproteins G2 and G1 could be synthesized and correctly processed in the absence of the 78- and 14-kDa proteins, as well as a large portion of the preglycoprotein sequences. However, the hydrophobic amino acid sequence immediately preceding the mature glycoprotein coding sequences was required for authentic glycoprotein production. The M-segment expression strategy involving aspects of translational initiation and protein processing are discussed. The functional roles of the 78- and 14-kDa proteins remain unclear.
    Journal of Virology 04/1988; 62(3):826-33. · 5.08 Impact Factor
  • Marc S Collett, Laura Torborg Kakach, Joann A. Suzich, Terri Wasmoen
    Virus Research. 11:22.