Kenneth A. Cornell

Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism.

The importance of methylthioadenosine/S‐adenosylhomocysteine (MTA/SAH) nucleosidase in bacteria has started to be appreciated only in the past decade. A comprehensive analysis of its various roles here demonstrates that it is an integral component of the activated methyl cycle, which recycles adenine and methionine through S‐adenosylmethionine (SAM)‐mediated methylation reactions, and also produces the universal quorum‐sensing signal, autoinducer‐2 (AI‐2). SAM is also essential for synthesis of polyamines, N‐acylhomoserine lactone (autoinducer‐1), and production of vitamins and other biomolecules formed by SAM radical reactions. MTA, SAH and 5′‐deoxyadenosine (5′dADO) are product inhibitors of these reactions, and are substrates of MTA/SAH nucleosidase, underscoring its importance in a wide array of metabolic reactions. Inhibition of this enzyme by certain substrate analogues also limits synthesis of autoinducers and hence causes reduction in biofilm formation and may attenuate virulence. Interestingly, the inhibitors of MTA/SAH nucleosidase are very effective against the Lyme disease causing spirochaete, Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes. These results indicate that inhibition of this enzyme can affect growth of different bacteria by affecting different mechanisms. Therefore, new inhibitors are currently being explored for development of potential novel broad‐spectrum antimicrobials.

Nucleosomes and DNA bind to specific cell-surface molecules on murine cells and induce cytokine production

The molecular basis for the cellular interaction of DNA and nucleosomes and the physiological consequences of this binding were examined. Both DNA and nucleosomes were demonstrated to bind specifically to the surface of human peripheral blood mononuclear cells and the murine T cell line S49. Western blots of S49 cell membranes, using probes of biotin-labeled DNA and nucleosomes, showed reactivity at 29 and 69 kDa. Functionally, the interaction of DNA and nucleosomes with murine spleen cells stimulated the release of significant amounts of IL-6 activity. There is evidence that nucleosomes, a product of apoptosis, are the major component of circulating DNA found in the plasma of patients with systemic lupus erythematosus (SLE). The interaction of nucleosomes with cell-surface DNA binding molecules may have physiological relevance to some of the immune aberrations observed in patients with SLE.

Suprabasal expression of human amphiregulin in the epidermis of transgenic mice induces a severe, early‐onset, psoriasis‐like skin pathology: Expression of amphiregulin in the basal epidermis is also associated with synovitis

Abstract:  The expression of amphiregulin (AR) in the basal epidermis of transgenic mice [keratin 14 promoter AR gene (K14‐ARGE)] has been previously shown to induce an early‐onset and severe skin pathology, with many similarities to psoriasis. In this study, it is demonstrated that involucrin enhancer/promoter‐dependent expression of human AR (INV‐AR) in the suprabasal epidermis of transgenic mice also produces a cutaneous psoriasis‐like phenotype. INV‐AR mice possess a limited lifespan and scaling, papillomatous, erythematous skin with partial alopecia. INV‐AR mouse histopathology also revealed epidermal hyperkeratosis, parakeratosis, acanthosis, and an exaggerated dermal vasculature. A dermal and epidermal infiltrate was also evident and consisted of both neutrophils and CD3+ T lymphocytes. The histology of synovial joints in both the INV‐AR mice and the K14‐ARGE mice of our previous investigation was examined. The histologic examination revealed that 3‐week‐old INV‐AR transgenic mice displayed normal knee joint histology, while 2‐ to 3‐week‐old K14‐ARGE transgenic mice frequently displayed synovitis, as exemplified by the presence of a mixed leukocytic infiltration, increased vascularization, and enhanced deposition of fibrous matrix in the knee synovium. These results demonstrate that AR overexpression in both the basal and suprabasal epidermis of transgenic mice induces a phenotype that mimics cutaneous psoriasis, while basal AR expression is also associated with synovial inflammation, a precursor to the psoriasis‐associated arthropathy, psoriatic arthritis. Collectively, the results implicate epidermal AR expression as a possible mediator of innate cutaneous immunity and epidermal proliferation and also as a potential trigger of both cutaneous psoriasis and psoriatic arthritis.

Functional Analysis of Methylthioribose Kinase Genes in Plants

Through a biochemical and a genetic approach, we have identified several plant genes encoding methylthioribose (MTR) kinase, an enzyme involved in recycling of methionine through the methylthioadenosine (MTA) cycle. OsMTK1, an MTR kinase from rice (Oryza sativa), is 48.6 kD in size and shows cooperative kinetics with a Vmax of 4.9 pmol/min and a K0.5 of 16.8 μm. MTR kinase genes are the first genes to be identified from the MTA cycle in plants. Insertional mutagenesis of the unique AtMTK gene in Arabidopsis (Arabidopsis thaliana) resulted in an inability of plants to grow on MTA as a supplemental sulfur source. MTK knock-out plants were not impaired in growth under standard conditions, indicating that the MTA cycle is a nonessential metabolic pathway in Arabidopsis when sulfur levels are replete. In rice, OsMTK genes were strongly up-regulated in shoots and roots when plants were exposed to sulfur starvation. Gene expression was largely unaffected by lack of nitrogen or iron in the nutrient solution, indicating that OsMTK regulation was linked specifically to sulfur metabolism.

Bgp, a secreted glycosaminoglycan-binding protein of Borrelia burgdorferi strain N40, displays nucleosidase activity and is not essential for infection of immunodeficient mice.

ABSTRACT Bgp, one of the surface-localized glycosaminoglycan-binding proteins of the Lyme disease spirochete, Borrelia burgdorferi, exhibited nucleosidase activity. Infection of SCID mice with B. burgdorferi strain N40 mutants harboring a targeted insertion in bgp and apparently retaining all endogenous plasmids revealed that Bgp is not essential for colonization of immunocompromised mice.

Autoimmunity to a 28–30 kD cell membrane DNA binding protein: occurrence in selected sera from patients with SLE and mixed connective tissue disease (MCTD)

Previous experiments have established the presence of a 30‐kD DNA binding protein on the surface of human leukocytes. Herein we report that selected sera from patients with systemic lupus erythematosus (SLE) and MCTD are reactive with a 28–30 kD protein on immunoblots of peripheral blood mononuclear cells (PBMC) cell membrane preparations; the reactivity is abolished by prior incubation of the blot with DNA. Antibodies ctuted from the 28–30 kD strip inhibited the binding of 3H.DNA to human PBMC. An immunomatrix of 28–30 kD reactive immunogtobulins was able to extract a 29‐kD DNA binding protein from a PBMC cell membrane preparation. Flow cytometry experiments confirmed the cell surface IgG reactivity of sera with T lymphocytes. Additional experiments indicated that cell surface IgG binding was not due to antibodies binding to cell surface DNA, DNA anti‐DNA immune complexes reacting with a DNA binding protein, anti‐histone antibodies or anti‐Sm antibodies. It is hypothesized that this autoimmune response could be one component of an idiotypic network involving anti‐DNA antibodies.

DNA Binding to Mouse Cells is Mediated by Cell-Surface Molecules: The Role of These DNA-Binding Molecules as Target Antigens in Murine Lupus

Autoimmunity to a 28-29-kDa cell-surface DNA-binding molecule has previously been described in patients with systemic lupus erythematosus and related autoimmune diseases. This report describes experiments that implicate a similar antigen-antibody system in the evolution of autoimmunity in lupus-prone mice. DNA binding to murine spleen cells was found to be a saturable phenomenon that was inhibited by excess cold DNA and trypsinization. The role of autoimmunity to murine cell-surface DNA-binding molecules in lupus-prone mice (MRL lpr/lpr, MRL + / +, BXSB) was compared to normal mice (BALB/c, C3H.SW) by means of an assay that measured the inhibition of cell-surface DNA binding. Only sera from lupus strains had inhibitory activity and this component was shown to be an IgM autoantibody. Furthermore, we isolated a spontaneously occurring IgM monoclonal antibody from the spleen of an MRL/lpr mouse, which inhibited DNA binding to mouse cells. Time-course studies indicated that young female MRL/lpr mice lacked detectable activity against cell- surface DNA-binding molecules; however, by 8-10 weeks maximal inhibitory activity was observed. This response occurred prior to the development of significant antinuclear antibody activity. With the appearance of overt disease and anti-DNA antibodies, inhibition of DNA- binding activity became undetectable. These findings mirror previous studies on autoimmunity to a cell-surface DNA-binding molecule on human leucocytes, but have the added advantage of permitting the study of the temporal evolution of this inhibitory activity in relation to disease expression.

DNA Vaccination Protects Mice against Challenge with Listeria monocytogenes Expressing the Hepatitis C Virus NS3 Protein

ABSTRACT The goal of this study was to develop a new surrogate challenge model for use in evaluating protective cell-mediated immune responses against hepatitis C virus (HCV) antigens. The use of recombinant Listeria monocytogenes organisms which express HCV antigens provides novel tools with which to assay such in vivo protection, as expression of immunity against this hepatotropic bacterial pathogen is dependent on antigen-specific CD8+ T lymphocytes. A plasmid DNA vaccine encoding a ubiquitin-NS3 fusion protein was generated, and its efficacy was confirmed by in vivo induction of NS3-specific, gamma interferon-secreting T cells following vaccination of BALB/c mice. These immunized mice also exhibited specific in vivo protection against subsequent challenge with a recombinant L. monocytogenes strain (TC-LNS3) expressing the NS3 protein. Notably, sublethal infection of naive mice with strain TC-LNS3 induced similar NS3-specific T-cell responses. These findings suggest that recombinant strains of L. monocytogenes expressing HCV antigens should prove useful for evaluating, or even inducing, protective immune responses against HCV antigens.

Hydroxy-anthraquinones as antimalarial agents

Abstract A series of hydroxy- and polyhydroxy-anthraquinones were screened for inhibitory activity against the malarial parasite, Plasmodium falciparum . Rufigallol demonstrated the most potent effects with a 50% inhibitory concentration (IC 50 value of ∼10.5 ng/ml (∼35 nM). Deleterious effects were exerted by rufigallol toward bone marrow progenitor cells at concentrations≥ 10 μM (3 μg/ml) where ∼30% suppression of colony growth was noted. Taking into consideration its potency and relative lack of toxicity, we believe that rufigallol should be advanced for in vivo studies. At the very least, rufigallol represents a simple, inexpensive lead drug for the development of more potent analogs.

Protection of interferon-γ knockout mice against Listeria monocytogenes challenge following intramuscular immunization with DNA vaccines encoding listeriolysin O

In this study we evaluated the efficacy of DNA vaccination of IFN-gamma knockout (GKO) mice against Listeria monocytogenes, as these immunodeficient mice are highly susceptible to infection with low numbers of this intracellular bacterial pathogen. Following intramuscular immunization of BALB/c GKO mice with plasmid DNA constructs encoding recombinant forms of the L. monocytogenes hemolysin, listeriolysin O (LLO), we detected the in vivo induction of a LLO(91-99) peptide-specific, protective immune CTL response equivalent to that observed following similar DNA vaccination of normal BALB/c mice. The observed protection represented greatly enhanced immunity for the GKO host, suggesting that DNA vaccination may provide a useful vaccine alternative for certain immunocompromised host populations.