(p)ppGpp-Dependent Regulation of the Nucleotide Hydrolase PpnN Confers Complement Resistance in Salmonella enterica Serovar Typhimurium.

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria, including Salmonella enterica serovar Typhimurium. S Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues, resulting in severe clinical outcomes. During infection, S Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5'-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane, known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

Streptococcal 5'-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion.

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 µm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.

SOBIR1 contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis.

The rate of entry of Magnaporthe oryzae into Arabidopsis pen2 sobir1 plants was significantly higher than that into pen2 plants. The length of the infection hyphae in pen2 sobir1 plants was significantly longer than that in pen2 plants. These results suggest that SOBIR1 is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.

Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs.

The hypersensitive response (HR) is a type of strong immune response found in plants that is accompanied by localized cell death. However, it is unclear how HR can block a broad range of pathogens with different infective modes. In this study, we report that γ-glutamylcysteine synthetase GSH1, which is critical for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to HR and block development of fungal pathogens with hemibiotrophic infective modes. We found that GSH1 is involved in the penetration2 (PEN2)-based entry control of the nonadapted hemibiotroph Colletotrichum gloeosporioides. However, Arabidopsis mutants specifically defective in entry control terminated further growth of the pathogen in the presence of HR cell death, whereas gsh1 mutants supported pathogen invasive growth in planta, demonstrating the requirement of GSH1 for postinvasive nonhost resistance. Remarkably, on the basis of the phenotypic and metabolic analysis of Arabidopsis mutants defective in Trp metabolism, we showed that biosynthesis of Trp-derived phytochemicals is also essential for resistance to C. gloeosporioides during postinvasive HR. By contrast, GSH1 and these metabolites are likely to be dispensable for the induction of cell death during postinvasive HR. Furthermore, the resistance to Ralstonia solanacearum 1/resistance to Pseudomonas syringae 4 dual Resistance gene-dependent immunity of Arabidopsis to the adapted hemibiotroph shared GSH1 and cytochromes P450 CYP79B2/CYP79B3 with postinvasive nonhost resistance, whereas resistance to P. syringae pv. maculicola 1 and resistance to P. syringae 2-based Resistance gene resistance against bacterial pathogens did not. These data suggest that the synthesis of glutathione and Trp-derived metabolites during HR play crucial roles in terminating the invasive growth of both nonadapted and adapted hemibiotrophs.

Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood.

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.

Phase I studies of peptide vaccine cocktails derived from GPC3, WDRPUH and NEIL3 for advanced hepatocellular carcinoma.

Aim: Two peptide cocktail vaccines using glypican-3, WD-repeat-containing protein up-regulated in hepatocellular carcinoma (HCC) and nei endonuclease VIII-like three epitopes were evaluated in advanced HCC in two Phase I studies. Patients & methods: Study 1 evaluated dose-limiting toxicities (DLTs) of peptides 1-3 (HLA-A24-restricted) and study 2 evaluated DLTs of peptides 1-6 (HLA-A24 or A02-restricted). Results: Overall, 18 and 14 patients were enrolled in studies 1 and 2, respectively. No DLTs were observed up to 7.1 mg of the vaccine cocktail. No complete response/partial response was observed. Stable disease was reported in nine and five patients with a disease control rate of 52.9% and 35.7% in studies 1 and 2, respectively. Conclusion: Both vaccines showed good tolerability and potential usefulness against HCC. Clinical trial registration: JapicCTI-121933; JapicCTI-142477.

Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of > 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However, this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols.

Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Production of a polyclonal antibody against inosine-uridine preferring nucleoside hydrolase of Acanthamoeba castellanii and its access to diagnosis of Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare disease but its prevalence throughout the globe continues to grow, primarily due to increased contact lens usage. Since early-stage symptoms associated with AK closely resemble those from other corneal infections, accurate diagnosis is difficult and this often results in delayed treatment and exacerbation of the disease, which can lead to permanent visual impairment. Accordingly, developing a rapid Acanthamoeba-specific diagnostic method is highly desired. In the present study, a rapid and differential method for AK diagnosis was developed using the secretory proteins derived from the pathogenic Acanthamoeba. Among the vast quantities of proteins secreted by the pathogenic Acanthamoeba, an open reading frame of the inosine-uridine preferring nucleoside hydrolase (IPNH) gene was obtained. After expressing and purifying the IPNH protein using the pGEX 4T-3 vector system, mice were immunized with the purified proteins for polyclonal antibody generation. Western blot was performed using protein lysates of the human corneal cell, non-pathogenic amoeba, pathogenic amoeba, and clinical amoeba isolate along with lysates from other causes of keratitis such as Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium solani to confirm Acanthamoeba-specificity. Western blot using the polyclonal IPNH antibody revealed that IPNH was Acanthamoeba-specific since these proteins were only observed in lysates of Acanthamoeba origin or its culture media. Our findings indicate that the IPNH antibody of Acanthamoeba may serve as a potential agent for rapid and differential AK diagnosis.

A combination DNA vaccine encoding nucleoside hydrolase 36 and glycoproteine 63 protects female but not male hamsters against Leishmania mexicana.

Leishmaniasis is a group of diseases caused by protozoan parasites of the Leishmania genus. Previous studies have shown that a DNA vaccine encoding Leishmania donovani antigen nucleoside hydrolase 36 and L. mexicana glycoprotein 63 is protective in mice. We investigated here the efficacy of this DNA vaccine to induce protection in golden hamsters. Male hamsters were more susceptible to infection by Leishmania mexicana than females. Following immunization with two doses of the DNA vaccine, only females resulted protected while males developed normal lesions.

Nucleoside Hydrolase NH 36: A Vital Enzyme for the Leishmania Genus in the Development of T-Cell Epitope Cross-Protective Vaccines.

NH36 is a vital enzyme of the DNA metabolism and a specific target for anti-Leishmania chemotherapy. We developed second-generation vaccines composed of the FML complex or its main native antigen, the NH36 nucleoside hydrolase of Leishmania (L.) donovani and saponin, and a DNA vaccine containing the NH36 gene. All these vaccines were effective in prophylaxis and treatment of mice and dog visceral leishmaniasis (VL). The FML-saponin vaccine became the first licensed veterinary vaccine against leishmaniasis (Leishmune®) which reduced the incidence of human and canine VL in endemic areas. The NH36, DNA or recombinant protein vaccines induced a Th1 CD4+IFN-γ+ mediated protection in mice. Efficacy against VL was mediated by a CD4+TNF-α T lymphocyte response against the NH36-F3 domain, while against tegumentary leishmaniasis (TL) a CD8+ T lymphocyte response to F1 was also required. These domains were 36-41 % more protective than NH36, and a recombinant F1F3 chimera was 21% stronger than the domains, promoting a 99.8% reduction of the parasite load. We also identified the most immunogenic NH36 domains and epitopes for PBMC of active human VL, cured or asymptomatic and DTH+ patients. Currently, the NH36 subunit recombinant vaccine is turning into a multi-epitope T cell synthetic vaccine against VL and TL.

NH36 and F3 Antigen-Primed Dendritic Cells Show Preserved Migrating Capabilities and CCR7 Expression and F3 Is Effective in Immunotherapy of Visceral Leishmaniasis.

Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.

Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections.

Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-gamma production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-gamma production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-gamma in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.

Protection of susceptible BALB/c mice from challenge with Leishmania major by nucleoside hydrolase, a soluble exo-antigen of Leishmania.

Leishmania major culture-derived, soluble, exogenous antigens have been shown to be a source of vaccine targets for the parasite. We have previously reported that L. major culture-derived, soluble, exogenous antigens can immunize BALB/c mice against challenge with L. major. However, the molecule(s) involved in this protection was not known. We describe the potential of one component of soluble exogenous antigens (recombinant nucleoside hydrolase) to vaccinate mice against challenge with L. major. We found that recombinant nucleoside hydrolase vaccinated BALB/c mice against a subsequent challenge with L. major. Protection was manifested by a significant decrease in lesion size (as much as a 30-fold reduction) and parasite burden (as much as a 71-fold reduction). Protection was achieved whether recombinant nucleoside hydrolase was administered to mice in the presence or absence of adjuvant (interleukin-12). Finally, protection was accompanied by an increase in interferon-gamma production but a decrease in interleukin-10 production by vaccinated animals in response to challenge with L. major.

Prediction and analysis of promiscuous T cell-epitopes derived from the vaccine candidate antigens of Leishmania donovani binding to MHC class-II alleles using in silico approach.

Visceral leishmaniasis is a dreadful infectious disease and caused by the intracellular protozoan parasites, Leishmania donovani and Leishmania infantum. Despite extensive efforts for developing effective prophylactic vaccine, still no vaccine is available against leishmaniasis. However, advancement in immunoinformatics methods generated new dimension in peptide based vaccine development. The present study was aimed to identify T-cell epitopes from the vaccine candidate antigens like Lipophosphogylcan-3(LPG-3) and Nucleoside hydrolase (NH) from the L. donovani using in silico methods. Available best tools were used for the identification of promiscuous peptides for MHC class-II alleles. A total of 34 promiscuous peptides from LPG-3, 3 from NH were identified on the basis of their 100% binding affinity towards all six HLA alleles, taken in this study. These peptides were further checked computationally to know their IFN-γ and IL4 inducing potential and nine peptides were identified. Peptide binding interactions with predominant HLA alleles were done by docking. Out of nine docked promiscuous peptides, only two peptides (QESRILRVIKKKLVR, RILRVIKKKLVRKTL), from LPG-3 and one peptide (FDKFWCLVIDALKRI) from NH showed lowest binding energy with all six alleles. These promiscuous T-cell epitopes were predicted on the basis of their antigenicity, hydrophobicity, potential immune response and docking scores. The immunogenicity of predicted promiscuous peptides might be used for subunit vaccine development with immune-modulating adjuvants.

Mammalian Ogg1/Mmh gene plays a major role in repair of the 8-hydroxyguanine lesion in DNA.

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cerevisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions. Recently, cDNA clones of four isoforms (types 1a, 1b, 1c, and type 2) of human OGG1 homologs (hMMH) were isolated. However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells. We have chosen two approaches to clarify this issue. First, using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we found that hMMH type 1a protein is in fact expressed in many types of human cells, showing that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity. Furthermore, we have shown that upon antibody-mediated depletion of hMMH type 1a protein in a whole-cell extract, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesion in human cells. In our second approach we have generated a mouse line carrying a mutant Mmh allele by targeted gene disruption. Mmh homozygous mutant mice were found to be physically normal in appearance, but to have lost the nicking activity for substrate DNA containing 8-OH-G in liver extracts. In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mutant mice at 8 weeks of age was 3-fold higher compared with wild-type or heterozygous mice. A further increase of 8-OH-G up to 7-fold was observed in 14-week-old animals. These results indicate that exposure of DNA to internal oxidative species constantly produces the mutagenic DNA adduct 8-OH-G in mice, and that Mmh plays an essential role in the repair of this type of oxidative DNA damage.

Biosynthesis of the human base excision repair enzyme uracil-DNA glycosylase.

The biosynthesis of the human DNA repair enzyme uracil-DNA glycosylase has been characterized by the reaction of in vitro- and in vivo-produced protein with an anti-human placental uracil-DNA glycosylase monoclonal antibody. In vitro synthesis of the DNA repair enzyme was examined after the translation of human placental polyadenylated [poly(A)+] RNA by immunoprecipitation of the [35S]methionine-labeled translation products. As defined by sucrose density analysis, immunoprecipitable in vitro products were translated from 16S poly(A)+ RNA and 11S poly(A)+ RNA. While the products of the 11S poly(A)+ RNA were smaller than purified uracil-DNA glycosylase, the product of the 16 S poly(A)+ RNA had a molecular weight of 37,000, identical to the size previously observed for purified human placental uracil-DNA glycosylase. Immunoblot analysis of human placental cell extracts and of normal human fibroblast cell extracts demonstrated the recognition of one Mr 37,000 protein. Immunoprecipitation of [35S]methionine-labeled normal human cell extracts with the anti-glycosylase monoclonal antibody specifically detected only the Mr 37,000 uracil-DNA glycosylase protein. Pulse-chase analysis demonstrated that the 35S radioactivity in the Mr 37,000 uracil-DNA glycosylase decreased over a 5-h interval. These results show that immunoreactive human uracil-DNA glycosylase protein was synthesized at its enzymatically active molecular weight of 37,000 as the primary translation product of a 16S polyadenylated messenger RNA.

Genetic cause of immune dysregulation - one gene or two?

Some autoimmune disorders are monogenetic diseases; however, clinical manifestations among individuals vary, despite the presence of identical mutations in the disease-causing gene. In this issue of the JCI, Massaad and colleagues characterized a seemingly monogenic autoimmune disorder in a family that was linked to homozygous loss-of-function mutations in the gene encoding the endonuclease Nei endonuclease VIII-like 3 (NEIL3), which has not been previously associated with autoimmunity. The identification of an unrelated healthy individual with the same homozygous mutation spurred more in-depth analysis of the data and revealed the presence of a second mutation in a known autoimmune-associated gene. Animals lacking Neil3 had no overt phenotype, but were predisposed to autoantibody production and nephritis following exposure to the TLR3 ligand poly(I:C). Together, these results support further evaluation of the drivers of autoimmunity in supposedly monogenic disorders.