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Organic semiconductors, including graphitic carbon nitride (g-C3N4, CN), represent an important class of materials for the development of novel antimicrobial or biomedical technologies. Of principal interest is the ability of these materials to catalyze the reduction of elemental oxygen to generate reactive oxygen species (ROS), including hydrogen peroxide (H2O2). Here, we describe the fabrication of photoactive van der Waals heterojunctions incorporating 1,4,5,8-naphthalene tetracarboxylic dianhydride (NTCDA) and CN. The composite heterojunction systems were characterized by a combination of physical (TEM, SEM, pXRD), spectroscopic (FT-IR, XPS, DRUV, photoluminescence, TCSPC) and kinetic experiments. Electronic interactions between the two components of the heterojunction increase the rate of photochemical production of H2O2 from elemental oxygen by 410%, relative to samples of pure CN. Mechanistic analysis reveals that interaction of NTCDA with the surface of CN modifies the mechanism of H2O2 formation in the heterojunction photocatalysts. The photochemical production of H2O2 by irradiation of the most active heterojunction composition is sufficient to reduce the viability of E. coli O157:H7, S. aureus and Ps. aeruginosa PAO1 by 99%. Importantly, H2O2 production by the NTCDA/CN heterojunctions suppresses Ps. aeruginosa biofilm formation, even at light exposure doses that had a lesser impact on overall planktonic cell growth.

Free-standing, composite hydrogels containing the visible-light responsive metal-free semiconductor graphitic carbon nitride (g-C3N4) as an integral component have been fabricated by direct casting...

Cold atmospheric pressure plasma (CAP) treatment has been shown to kill bacteria and remove bacterial biofilms from surfaces. Here we report the etch capacity of a linear discharge CAP device on Pseudomonas fluorescens biofilms. A 21 kHz, 1.4 kV RMS AC voltage applied to the CAP electrodes generated a hydrated Ar plasma between the plates, with the gas flow directing the plasma species toward the biological sample, causing both bacterial killing and etching of the biofilm. Typical discharge currents for a 2.4 cm long, 0.6 mm wide linear discharge device were 1–4.4 mA. Hydrated Ar flow gas was critical for removal of biofilm from a stainless steel substrate, while both hydrated and dry Ar + O2, Ar + air, O2 only, and air only flow gas mixtures did not cause etching at equivalent or greater discharge current intensities. A biofilm etch rate of > 2 μm/min was achieved, provided the plasma discharge was within 1–2 mm of the substrate surface and used a hydrated Ar gas flow of at least 5 LPM.

Organic semiconductors, including graphitic carbon nitride (g-C

Cold atmospheric-pressure plasma (CAP) has been shown to kill bacteria and remove biofilms. Here, we report the development of a unique CAP array device consisting of a parallel stack of eight linear-discharge plasma elements that create a $\sim 5$ -cm $^{\mathbf {2}}$ (2.4 cm $\times 2$ cm) treatment area. The CAP device is fabricated from low-temperature cofired ceramic (LTCC) layers to create 24-mm-long linear-discharge channels (500- $\mu \text{m}$ gap) with embedded opposing silver metal electrodes. A 20-kHz ac voltage (0.5–5 kV) applied to the electrodes generates an Ar/ $\text{O}_{\mathbf {2}}$ plasma between the plates, with the gas flow directing the reactive species toward the biological sample (biofilms and so on) to affect the antimicrobial treatment. External ballast resistors were used to study discharge uniformity in the stacked array elements, and internal thick film ballast resistors ( $\approx 150~\text{k}\Omega $ ) were developed to create a fully integrated device. Typical element discharge currents were 1–2.5 mA with the total array current tested at 20 mA to provide optimal device uniformity. The plasma discharge was further shown to produce reactive hydrogen peroxide and exert antimicrobial effects on Pseudomonas biofilms and Salmonella contaminated eggshell samples, with >99% of the bacterial cells killed with less than 60 s of plasma exposure.

The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5&prime, deoxy-5&prime, iodoadenosine (IADO) and 5&prime, ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease.

In this manuscript, we describe the fabrication of photoactive biocidal or sporicidal films from urea-derived graphitic carbon nitride (u-g-C3N4). Co-deposited films of u-g-C3N4 and Escherichia coli O157:H7 (IC50 = 14.1 ± 0.2 mJ) or Staphylococcus aureus (methicillin resistant IC50 = 33.5 ± 0.2 mJ, methicillin sensitive IC50 = 42.7 ± 0.5 mJ) demonstrated significantly enhanced bactericidal behavior upon administration of visible radiation (400 nm ≤ λ ≤ 426 nm). In all cases, complete eradication of the microbial sample was realized upon administration of 100 mJ of visible radiation, while no antimicrobial activity was observed for non-irradiated samples. In contrast, Bacillus anthracis endospores were more resistant to u-g-C3N4 mediated killing with only a ca. 25% reduction in spore viability when treated with a 200 mJ dose of visible radiation. Characterization of u-g-C3N4 reveals that the improved activity results from enhancements of both the surface area and reduction potential of the material’s conduction band edge, coupled with fast injection of charge carriers into localized states and a decline in radiative recombination events. The results of this study demonstrate that g-C3N4-based materials offer a viable scaffold for the development of new, visible light driven technologies for controlling potentially pathogenic microorganisms.

Photoactive films derived from nanostructured samples of metal-free, intermediate band gap semiconductor graphitic carbon nitride (ns-g-C3N4) have been synthesized and characterized for their particle properties and antimicrobial activity. Physical characterization reveals that these materials are composed of discrete nanoparticles whose dimensions range from 200 nm to 700 nm. Investigation of the photochemical reactivity of ns-g-C3N4 using coumarin-3-carboxylic acid (3-CCA) indicates that this material produces reactive oxygen species (ROS) under visible radiation. When irradiated with 0.31 J visible light, ns-g-C3N4-based materials reduced the viability of both Gram-negative Escherichia coli O157:H7 and Gram-positive Staphylococcus aureus by approximately 50%. Nearly complete inactivation of both strains of microorganisms was achieved upon administration of a 0.62 J dose of visible radiation. Importantly, no biocidal activity was observed for non-irradiated samples, indicating that the g-C3N4-derived films are not inherently toxic in the absence of visible light. The results of this study suggest that materials and, by extention, films and coatings derived from g-C3N4 may present a novel route for controlling pathogenic microorganisms on surfaces in the environment, and could be useful in reducing incidents of hospital-acquired infections.

Two synthetic aziridinomitosenes (AZMs), Me-AZM and H-AZM, structurally related to mitomycin C (MC) were evaluated for their anticancer activity against six cancer cell lines (HeLa, Jurkat, T47D, HepG2, HL-60, and HuT-78) and tested for their DNA-modifying abilities in Jurkat cells. Cytotoxicity assays showed that Me-AZM is up to 72-fold and 520-fold more potent than MC and H-AZM, respectively. Me-AZM also demonstrated increased DNA modification over MC and H-AZM in alkaline COMET and Hoechst fluorescence assays that measured crosslinks in cellular DNA. Me-AZM and H-AZM treatment of Jurkat cells was found to sponsor significant DNA-protein crosslinks using a K-SDS assay. The results clearly indicate that the AZM C6/C7 substitution pattern plays an important role in drug activity and supports both DNA-DNA and DNA-protein adduct formation as mechanisms for inducing cytotoxic effects.

In this manuscript, we describe the fabrication of photoactive biocidal or sporicidal films from urea-derived graphitic carbon nitride (u-g-C3N4). Co-deposited films of u-g-C3N4 and Escherichia coli O157:H7 (IC50 = 14.1 ± 0.2 mJ) or Staphylococcus aureus (methicillin resistant IC50 = 33.5 ± 0.2 mJ, methicillin sensitive IC50 = 42.7 ± 0.5 mJ) demonstrated significantly enhanced bactericidal behavior upon administration of visible radiation (400 nm ≤ λ ≤ 426 nm). In all cases, complete eradication of the microbial sample was realized upon administration of 100 mJ of visible radiation, while no antimicrobial activity was observed for non-irradiated samples. In contrast, Bacillus anthracis endospores were more resistant to u-g-C3N4 mediated killing with only a ca. 25% reduction in spore viability when treated with a 200 mJ dose of visible radiation. Characterization of u-g-C3N4 reveals that the improved activity results from enhancements of both the surface area and reduction potential of the material’s conduction band edge, coupled with fast injection of charge carriers into localized states and a decline in radiative recombination events. The results of this study demonstrate that g-C3N4-based materials offer a viable scaffold for the development of new, visible light driven technologies for controlling potentially pathogenic microorganisms.

Background Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. Methods Here we examine three unique 5′-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. Results The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5′-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5′-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3–0.4 μg/mL against cultured B. burgdorferi cells. Conclusion B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. General significance The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.

The increasing use of low temperature co-fired ceramic (LTCC) for the fabrication of biological microfluidic devices necessitates further research on LTCC biocompatibility. In this study we explore the inhibitory effect of DuPont's 951 LTCC on Polymerase Chain Reaction (PCR), and demonstrate a novel mechanism to increase biocompatibility between LTCC and PCR with the addition of a common passivation substance, bovine serum albumin (BSA). We show that DuPont's 951 LTCC binds negatively charged proteins including BSA and ovalbumin (OVA). This is a significant discovery as proteins (enzymes) are an essential component of most biological reactions, and a frequent addition to microfluidic devices. A proposed model for LTCC inhibition of PCR by enzyme adsorption is presented.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimental liver fibrosis. To elicit fibrosis, C57BL6/ male mice were treated twice weekly for 8 weeks with 0.5 ml/kg carbon tetrachloride (CCl4). TCDD (20 μg/kg) or peanut oil (vehicle) was administered once a week during the last 2 weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl4-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homologue-1, TGF-β1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl4-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling.

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.

Background: Lyme disease is the most prevalent tick-borne disease in the USA with the highest number of cases (27444 patients) reported by CDC in the year 2007, representing an unprecedented 37% increase from the previous year. The haematogenous spread of Borrelia burgdorferi to various tissues results in multisystemic disease affecting the heart, joints, skin, musculoskeletal and nervous system of the patients. Objectives: Although Lyme disease can be effectively treated with doxycycline, amoxicillin and cefuroxime axetil, discovery of novel drugs will benefit the patients intolerant to these drugs and potentially those suffering from chronic Lyme disease that is refractory to these agents and to macrolides. In this study, we have explored 5 0 -methylthioadenosine/S-adenosylhomocysteine nucleosidase as a drug target for B. burgdorferi, which uniquely possesses three genes expressing homologous enzymes with two of these proteins apparently exported. Methods: The recombinant B. burgdorferi Bgp and Pfs proteins were first used for the kinetic analysis of enzymatic activity with both substrates and with four inhibitors. We then determined the antispirochaetal activity of these compounds using a novel technique. The method involved detection of the live‐dead B. burgdorferi by fluorometric analysis after staining with a fluorescent nucleic acids stain mixture containing Hoechst 33342 and Sytox Green. Results: Our results indicate that this method can be used for high-throughput screening of novel antimicrobials against bacteria. The inhibitors formycin A and 5 0 -p-nitrophenythioadenosine particularly affected B. burgdorferi adversely on prolonged treatment. Conclusions: On the basis of our analysis, we expect that structure-based modification of the inhibitors can be employed to develop highly effective novel antibiotics against Lyme spirochaetes.

5'-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5'-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5'-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 A resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5'-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5'-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK(a) of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.

The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp{sup 250}-Glu{sup 252}). In addition, the glycine-rich loop of the protein, analogous to the 'Gly triad' in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp{sup 233} of the catalytic HGD motif, a novel twin arginine motif (Arg{sup 340}/Arg{sup 341}), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosylmore » moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.« less

Methylthioadenosine (MTA) is released as a by-product of S-adenosylmethionine (AdoMet)-dependent reactions central to ethylene, polyamine, or phytosiderophore biosynthesis. MTA is hydrolysed by methylthioadenosine nucleosidase (MTN; EC 3.2.2.16) into adenine and methylthioribose which is processed through the methionine (Met) cycle to produce a new molecule of AdoMet. In deepwater rice, submergence enhances ethylene biosynthesis, and ethylene in turn influences the methionine cycle through positive feedback regulation of the acireductone dioxygenase gene OsARD1. In rice, MTN is encoded by a single gene designated OsMTN. Recombinant OsMTN enzyme had a K M for MTA of 2.1 mM and accepted a wide array of 5′ substitutions of the substrate. OsMTN also metabolized S-adenosylhomocysteine (AdoHcy) with 15.9% the rate of MTA. OsMTN transcripts and OsMTN-specific activity increased slowly and in parallel upon submergence, indicating that regulation occurred mainly at the transcriptional level. Neither ethylene, MTA, nor Met regulated OsMTN expression. Analysis of steady-state metabolite levels showed that MTN activity was sufficiently high to prevent Met and AdoMet depletion during long-term ethylene biosynthesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-β. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1′ bond in the nucleosides, 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant–enzyme and mutant–product complexes have been determined at 2.0 A, 2.0 A, and 2.1 A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR·ADE structures are the first enzyme–substrate and enzyme–product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A “catalytic movie” detailing substrate binding, catalysis, and product release is presented.

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.

Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria. It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2). We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling. Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay. The chemical entities of products from the LuxS reaction were also established. Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.

In this study we evaluated the efficacy of DNA vaccination of IFN-γ 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.

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical biological processes in many microbes. This nucleosidase catalyzes the irreversible hydrolysis of the N(9)-C(1') bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have been solved to 2.2- and 2.0-A resolution, respectively. These are the first MTA/AdoHcy nucleosidase structures to be solved in the presence of inhibitors. These structures clearly identify the residues involved in substrate binding and catalysis in the active site. Comparisons of the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase (Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced conformational change in the active site and the substrate preference of the enzyme. The enzymatic mechanism has been re-examined.

The development of protective immunity against many intracellular bacterial pathogens commonly requires sublethal infection with viable forms of the bacteria. Such infection results in the in vivo activation of specific cell-mediated immune responses, and both CD4+ and CD8+ T lymphocytes may function in the induction of this protective immunity. In rodent models of experimental infection with Listeria monocytogenes, the expression of protective immunity can be mediated solely by the immune CD8+ T cell subset. One major target Ag of Listeria-immune CD8+ T cells is the secreted bacterial hemolysin, listeriolysin O (LLO). In an attempt to generate a subunit vaccine in this experimental disease model, eukaryotic plasmid DNA expression vectors containing genes encoding either the wild-type or modified forms of recombinant LLO were generated and used for genetic vaccination of naive mice. Results of these studies indicate that the intramuscular immunization of mice with specifically designed plasmid DNA constructs encoding recombinant forms of LLO stimulates peptide-specific CD8+ immune T cells that exhibit in vitro cytotoxic activity. More importantly, such immunization can provide protective immunity against a subsequent challenge with viable L. monocytogenes, demonstrating that this experimental approach may have direct application in prevention of acute disease caused by intracellular bacterial pathogens.

In a previous report we described the synergistic antimalarial interaction between two structurally similar compounds, rufigallol and exifone. To explain this phenomenon, we proposed that exifone is transformed inside the parasitized erythrocyte into a xanthone with potent antimalarial properties. We speculated that the transformation process was induced by the prooxidant activity of rufigallol. On the basis of this model we hypothesized that exifone would act synergistically with other oxidant drugs. In the present study we have found a similar synergistic interaction between exifone and ascorbic acid (vitamin C) against both chloroquine-susceptible and multidrug-resistant strains of Plasmodium falciparum. The prooxidant activity of ascorbic acid against Plasmodium-infected erythrocytes is believed to result from an intraerythrocytic Fenton reaction occurring in the acidic food vacuole of the parasite. The hydroxyl radicals produced during this process are believed to attack exifone, which undergoes cyclodehydration to become 2,3,4,5,6-pentahydroxyxanthone (X5). Evidence presented to support this "xanthone hypothesis" includes the demonstration that the exifone ==> X5 transformation occurs readily in vitro under mildly acidic conditions in the presence of iron, ascorbic acid, and oxygen.

Recombinant E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) was used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention. An examination of the parameters required for enzymatic activity indicate that the nucleosidase functions over a broad range of pH and temperature, with acidic conditions and temperatures of 37-45 degrees C being optimal. Analogs of 5'-methylthioadenosine and adenosine were assessed as potential enzyme inhibitors and to provide details regarding substrate specificity and reaction mechanism. The 5'-arylthio analog, 5'-(p-nitrophenyl)thioadenosine, was the most potent enzyme inhibitor studied, with a Ki of 20nM. A mutant of the nucleosidase lacking the first 8 amino acids was engineered to determine the contribution of these conserved residues toward enzyme specificity. The truncated enzyme exhibited a K(m)[MTA] of 1.43 microM, approximately 3 fold higher than the K(m) reported for the full-length nucleosidase.

We have discovered a remarkable synergistic antimalarial interaction between rufigallol and the structurally similar compound exifone. The synergistic effects were produced in chloroquine-susceptible and chloroquine-resistant clones of Plasmodium falciparum. The degree of potentiation as estimated by standard isobolar analysis was approximately 60-fold for experiments initiated with asynchronous parasites. The most pronounced synergism was observed in experiments with synchronized trophozoite-infected erythrocytes, in which the degree of synergy was at least 300-fold. While the mechanism underlying this drug potentiation remains unresolved, it is hypothesized that rufigallol acts in pro-oxidant fashion to produce oxygen radicals inside parasitized erythrocytes. These radicals would attack exifone, thereby initiating its transformation into a more potent compound, a xanthone.

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.

Halogen substituted analogs of 5-(phenylthio)ribose were synthesized and tested as inhibitors of the methylthioribose kinase-containing pathogen. Klebsiella pneumoniae . All of the compounds exhibit good activity against organism. 5-(4-Iodophenylthio)ribose demonstrated the most potent inhibitory effects with a 50% inhibitory concentration (IC 50 ) value of ′ 2.5 picomolar (0.0009 ng/ml).

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

A recombinant form of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (E.C. 3.2.2.9) has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. While several different crystallization conditions were obtained, only one set of conditions yielded crystals suitable for X-ray diffraction analysis. These crystals grow as diamond-shaped wedges, with unit-cell parameters a = 50.92, b = 133.99, c = 70.88 A, alpha = beta = gamma = 90 degrees. The crystals belong to space group P2(1)2(1)2 and diffract to a minimum d spacing of 2.3 A on a MAR345 image plate with a Rigaku RU-200 rotating-anode X-ray generator. On the basis of density calculations, two monomers are predicted per asymmetric unit (Matthews coefficient, V(M) = 2.37 A(3) Da(-1)), with a solvent content of 48%.

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.

SUMMARYAnti-idiotypic anti-DNA antibodies (anti-anti-DNA) have previously been described in both patients with systemic lupus erythematosus and healthy individuals. Jerne's hypothesis predicts that such antibodies would bear a paratope reactive with non-sequence specific DNA binding proteins. Here we have explored the notion of a molecular mimicry between anli-anti-DNA antibodies and antibodies to a previously described 28–29 kD cell surface DNA binding molecule. It was shown that affinity purified anti-anti-DNA antibodies inhibit the binding of DNA to cells and that MoAb to the 28–29 kD receptor react with anti-DNA antibodies. These findings indicate that a subset of anti-anti-DNA antibodies are idiotypically related to antibodies reactive with a cell surface DNA binding molecule. It is hypothesized that anti-DNA antibodies may arise when a convergence of genetic and environmental influences favours an unrestrained anti-idiotypic response to cell surface DNA binding molecule(s).

Parkinson's disease is a debilitating movement disorder characterized by altered levels of alpha(6)beta(2) * ( * indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. alpha-Conotoxin MII (alpha-CTx MII) is a highly useful ligand to probe alpha(6)beta(2) nAChRs structure and function, but it does not discriminate among closely related alpha(6) * nAChR subtypes. Modification of the alpha-CTx MII primary sequence led to the identification of alpha-CTx MII[E11A], an analog with 500-5300-fold discrimination between alpha(6) * subtypes found in both human and non-human primates. alpha-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity alpha(6) nAChR, a subtype that is selectively lost in Parkinson's disease. Here, we present the three-dimensional solution structure for alpha-CTx MII[E11A] as determined by two-dimensional (1)H NMR spectroscopy to 0.13+/-0.09A backbone and 0.45+/-0.08A heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of alpha-CTx MII[E11A] relative to other members of the alpha-CTx family may be responsible for its exceptionally high affinity for alpha6alpha4beta2 * nAChR as well as discrimination between alpha(6)beta(2) and alpha(3)beta(2) containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for alpha(6) * nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson's disease.

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.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.

Immucillin and DADMe-Immucillin inhibitors are tight binding transition state mimics of purine nucleoside phosphorylases (PNP). 5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is proposed to form a similar transition state structure as PNP. The companion paper describes modifications of the Immucillin and DADMe-Immucillin inhibitors to better match transition state features of MTAN and have led to 5′-thio aromatic substitutions that extend the inhibition constants to the femtomolar range (Singh, V., Evans, G. B., Lenz, D. H., Mason, J., Clinch, K., Mee, S., Painter, G. F., Tyler, P. C., Furneaux, R. H., Lee, J. E., Howell, P. L., and Schramm, V. L. (2005) J. Biol. Chem. 280, 18265-18273). 5′-Methylthio-Immucillin A (MT-ImmA) and 5′-methylthio-DADMe-Immucillin A (MT-DADMe-ImmA) exhibit slow-onset inhibition with Ki* of 77 and 2 pm, respectively, and were selected for structural analysis as the parent compounds of each class of transition state analogue. The crystal structures of Escherichia coli MTAN complexed with MT-ImmA and MT-DADMe-ImmA were determined to 2.2 A resolution and compared with the existing MTAN inhibitor complexes. These MTAN-transition state complexes are among the tightest binding enzyme-ligand complexes ever described and analysis of their mode of binding provides extraordinary insight into the structural basis for their affinity. The MTAN-MT-ImmA complex reveals the presence of a new ion pair between the 4′-iminoribitol atom and the nucleophilic water (WAT3) that captures key features of the transition state. Similarly, in the MTAN-MT-DADMe-ImmA complex a favorable hydrogen bond or ion pair interaction between the cationic 1′-pyrrolidine atom and WAT3 is crucial for tight affinity. Distance analysis of the nucleophile and leaving group show that MT-ImmA is a mimic of an early transition state, while MT-DADMe-ImmA is a better mimic of the highly dissociated transition state of E. coli MTAN.

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.

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.

The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. 5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5'-methylthiotubercidin have been solved to 2.0 and 2.1 A resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5'-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5'-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.

Extracellular adenosine and its related nucleotides have been referred to as retaliatory metabolites that can be released into the extracellular environment during inflammation, wounding, and other pathologic states. We have previously reported that these compounds reversibly inhibit the proliferation of normal keratinocyte cultures and we now demonstrate that these compounds also arrest the proliferation of transformed keratinocytes. Although our study shows that keratinocytes express mRNA corresponding to the A2B purinoreceptors and that adenosine or AMP treatment elevates intracellular cAMP in these cells, our study also demonstrates that dipyridamole-inhibitable transport of adenosine into the keratinocyte is central to the mechanism by which adenosine and adenine nucleotides arrest proliferation in these cells. In support of this mechanism, our results demonstrate that human keratinocytes express mRNA corresponding to the recently cloned dipyridamole-sensitive human equilibrative nucleoside transporter. Interestingly, coincubation with adenosine deaminase reverses the antiproliferative action of adenosine and exerts no effect on the antiproliferative activity of the adenine nucleotides, thus supporting a model in which adenine nucleotides are enzymatically converted to adenosine and transported into the keratinocyte in a tightly coupled and adenosine-deaminase-resistant manner. Analysis of adenosine- and adenosine-monophosphate-treated keratinocytes demonstrated that quiescence is induced within 12–24 h, and fluorescence-activated cell sorter analysis suggests that treatment with these compounds may result in the inhibition of keratinocyte proliferation at both G1 and S phases of the cell cycle. In addition to their documented antiproliferative action on other cell types, adenosine, adenine nucleotides, and related analogs may also represent a potential new class of pharmacologic regulators of keratinocyte proliferation in vivo

The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) is responsible for cleavage of the glycosidic bond in both 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). Based on amino acid sequence analysis of this enzyme from Klebsiella, we recently speculated that an open reading frame found in E. coli (designated pfs) encoded MTA/SAH nucleosidase. To explore this possibility, we amplified, cloned, and expressed the complete pfs gene from E. coli genomic DNA. The recombinant protein exhibited a molecular weight and Michaelis constants for MTA that are in agreement with those reported for native enzyme. From this biochemical evidence we confirm our original assignment of the pfs gene as encoding MTA/SAH nucleosidase.

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.

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.